PONE-D-20-29560

Decreased Content of Ascorbic acid (Vitamin C) in the Brain of a Mouse Model of ATP1A3-related Neurologic Disorders

PLOS ONE

Dear Dr. Ikeda,

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.

Both reviewers showed great interests in the core findings of the manuscript that the mutant mice showed significant decreased ASC level. I also found the results fascinating and potentially opened a new avenue of research in the field. However, both reviewers also pointed out deficiencies of the manuscript that needs to be addressed before the manuscript can be published. Reviewer 1 requested further study of the heterozygous SVCT2 mice, in vitro study of SVCT2 cell culture, ouabain in vivo or cell culture experiment. Reviewer 1 also expressed concerns of off target effect of the CRISPR experiments. Reviewer 2 requested additional information on the expression levels of SVCT2. I do not see any conflicts between the comments of these two excellent reviewers. I believe these additional experiments will improve the manuscript if the animal and cell resources are readily available and can be completed in a timely manner.  

Please submit your revised manuscript by Nov 21 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Yuqing Li, Ph.D.

Academic Editor

PLOS ONE

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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: Partly

Reviewer #2: Yes

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

Reviewer #1: I Don't Know

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: Yes

Reviewer #2: Yes

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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: In this article Ikeda et al. compared the phenotypes of mouse fetuses with double homozygous knockout of Atp1a2 and Atp1a3 (α2α3-dKO) to those of fetuses with single knockout. They found:

1. The brain hemorrhage phenotype of α2α3-dKO was quite similar to that of fetuses with homozygous knockout of the gene encoding ascorbic acid (ASC) transporter SVCT2.

2. The α2α3-dKO brain showed significantly decreased levels of ASC compared with the single knockout mouse brain

3. ASC content in the basal ganglia and cerebellum was significantly lower in the adult Atp1a3 heterozygous knockout mouse (α3-HT) than in the wild-type mouse.

4. Significant decrease in ASC in the cerebellum of α3-HT mice in the peripartum period, during which mice are under physiological stress.

The authors then conclude that their observations indicate that the α2 and α3 subunits independently and additionally contribute to the ASC level in the fetal brain and that the α3 subunit contributes to the adult basal ganglia and cerebellum. They then propose that decreased ASC levels may affect neural network development and are linked to the pathophysiology of symptoms in ATP1A2 and related neurologic disorders.

Overall Comments:

This article generates intriguing data of very original hypotheses very worthy of investigation. However, it fails, with the experiments performed so far, to provide a coherent narrative supported by strong data. Substantially more work is needed to be able to support such a coherent narrative.

The demonstration of reduced levels of ASC does not necessarily indicate that this reduction is contributing significantly to the pathology or pathophysiology of ATP1A2 and ATP1A3 disease. To support this hypothesis of theirs they would need to demonstrate that such reductions in the absence of ATP1A2 and ATP1A3 mutations reproduce the phenotype and physiology of the heterozygous knock out mice. One way to do that is to study the heterozygous or some similar transgenic model of the ASC transporter determine the levels in such a model, demonstrate these are in the range of what they observed in their heterozygous knockouts and show phenotype and neurophysiological similarities. The comparisons of the homozygous severe phenotypes and double knockout, where as interesting, are generating severe phenotypes that may not be relevant to the actual disease model (heterozygous knock outs) or to the human condition. Of note, for example, is that patients with ATP1A3 disease have not been noted to have intracranial hemorrhage. Performing the above is important particularly that the authors did not find any decreases in malondialdehyde (MDA), and sulfhydryl (SH) which does not support their hypotheses and because they found that ASC levels were not decreased in cortex. It is known that mice and humans with ATP1A2 and ATP1A3 related disease do have cortical abnormalities. This argues against their hypotheses.

Additional experiments that also could support their hypotheses are in vitro studies to demonstrate in cell cultures the effect of the mutation on ASC transport.

Abstract and Introduction:

Those are too long and can be summarized.

Methods and study design:

The authors need to justify the relevance of why they used study double homozygous knockout mice if there is no human with this condition.

Which parts of the study were done in which university and what was the role of Adriana A. Tienda, and of Fiona E. Harrison

What were the Coefficient of variations and of the assays they performed.

What program was used for statistics?

The authors need to study the hippocampus, or justify why it was not studied, since in many studies it was shown that this structure has significant role in ATP1A3 mutant mice phenotype.

Results:

The authors did not show directly that Na/K pump is essential for ASC transporting. They have to use Na/K pump inhibitor (ouabain) or in vitro experiments in cell cultures to demonstrate this relationship.

Figure 3 indicated that the alpha 3 knockout adults have lower ASC levels than WT while figure 6 appears to indicate there is no difference. This needs to be clarified.

In Figure 1 there is no described sequence event though in they indicate in the text that the sequence is in that figure.

What is the role of ATP1A3 and ATP1A2 with abdominal wall rapture? Maybe there is off target mutation? How can the authors be sure that there is no off target mutation given that they used CRISPR technique? Sanger sequencing of targeted/investigated genes could be essential.

Regarding the statement “To examine whether physiological stress decreased brain vitamin C in ATP1A3 mouse model affects ASC levels in the brain, we measured ASC content in brain regions (cortex, basal ganglia, and cerebellum) of peripartum female mice that were either in full-term pregnancy or lactating within 12 hours of delivering a litter (Fig. 6).” Question regarding this experiments: How physiological stress performed? Or are they just referring to the stress of the peripartum period?

Discussion

The authors need to discuss the implications that in humans ATP1A2 non neuronal and emphasize even more that their double knock out mice do not model the human condition so they only raise the possibility that vitamin C may contribute to the pathophysiology of symptoms manifested in humans.

The discussion omits important implications and potential correlations with the human conditions and with the other related models that actually would indirectly support a possible relationship of ASC in ATP1A2 and ATP1A3 related disease. These need to be added. They include 1) ASC can block glutamatergic dysfunction (PMID: 7816935) and ATP1A2 and ATP1A3 disease models (PMID: 30071271 PMID: 19666602 PMID: 25523819 PMID: 27445835). 2) ASC enhances GABA function (PMID 21715633) and GABA dysfunction is prominent in ATP1A3 related disease models (PMID : 29889309). 3) ASC deficiency can produce congenital brain anomalies with neonatal manifestation (PMID: 25487414) and ATP1A2 and ATP1A3 related disease can manifest similarly in humans (PMID: 31608932, PMID: 32802951, PMID: 31484714). 4) There most remarkable findings were in the cerebellum, this is consistent with prior data about the importance of ASC in formation of the cerebellum (PMID: 4830116). It also consistent with data from human studies that shows that ATP1A3 related disease frequently shows congenital and subsequent cerebellar hypoplasia and atrophy (PMID: 24651393, PMID: 32115366, PMID: 31950366).

The statement “Since the post-birth deaths of these Decreased brain vitamin C in ATP1A3 mouse model (Page 25) mice were likely due to the lack of spontaneous respiratory neural activity, in addition to severe hemorrhage inside the brainstem, it is suggested that these mice exhibit defects in the respiratory neural network [37,47], similar to Na + , K+ -ATPase α2 and α3 homozygous knockout mice (α2N-KO and α3-KO)” is purely conjectural. They should provide data to support that. They did not measure neural activity so how do they know that their observation are due to lack of spontaneous respiratory neural activity, how do they know that this is not due to primary cardiac or epileptic activity causing secondary apnea?

Minor Points

Abbreviations need to be included at time of first mention of compound (e.g of malondialdehyde (MDA).

References are needed for the following statements: “Because many neurological diseases are characterized by decreased brain vitamin C (need reference(s)… in ATP1A3 mouse.”; And “Alternatively, the function of the α2 subunit may be compensated by the ubiquitously expressed α1 subunit during brain development”.

The quality of Figure 5 needs to be improved since some words are difficult to read.

Reviewer #2: The main finding reported in the manuscript is reduced levels of ascorbic acid (vitamin C) in NKA alpha2 KO, NKA alpha3 KO and NKA alpha2/alpha3 double KO whole foetal mouse brain, in addition to basal ganglia and cerebellum from 2- to 4-month-old adult NKA alpha3 heterozygous mice. This is a novel and interesting finding. The manuscript is generally well written and presented. I do have several minor points of criticism though, which prevent the manuscript from being suitable for publication in its current form.

1. The Introduction refers to heterozygous mutations in ATP1A2 and ATP1A3 that are related to human neurological disorders, but the main types of mutation involved are not specified. Given the subjects of the study, readers might assume that deletions or nonsense mutations of ATP1A2 and ATP1A3 were the primary cause of the disorders mentioned, but this is not correct. The authors should clarify that it is actually missense mutations that are the primary cause.

2. On page 95, the authors should clarify what is meant by "homozygous missense/truncating (deletion)". Missense mutations are different from truncating mutations, which could be nonsense mutations.

3. In the Materials and Methods and in Table 1, the authors should clarify the identities of the mouse lines used. Specifically, it would be helpful if they used the official Mouse Genome Informatics nomenclature, at least once, as follows:

http://www.informatics.jax.org/allele/MGI:2664907

Atp1a2tm1Kwk

Atp1a2-, C-KO

http://www.informatics.jax.org/allele/MGI:3522421

Atp1a2tm2Kwk

N-KO

http://www.informatics.jax.org/allele/MGI:5572809

Atp1a3tm1.1Kwk

4. In the Materials and Methods, the diet fed to the mice is described merely as "Food (a standard pelleted rodent diet containing 4 mg ASC in 100 g pellet)". This is an inadequate description. The composition of the mouse diet should be stated more thoroughly.

5. In the sub-section headed Creation of Slc23a2-KO mice, the authors should rephrase and clarify the following sentence, as it is not clear to this reviewer: "Because all the latter were found to be random integrations in non-homologous chromosomes, we used only Slc23a2-KO for the subsequent analyses."

6. In the Results, the authors focus on sodium-dependent vitamin C transporter 2 (SVCT2), encoded by Slc23a2. It is not shown whether NKA alpha2 KO, NKA alpha3 KO and NKA alpha2/alpha3 double KO whole foetal mouse brain has wild-type like levels of SVCT2. It would be helpful if the authors used IHC, western blotting or RT-PCR to examine the levels of SVCT2 or Slc23a2 in whole foetal brain from these mouse lines. If levels of SVCT2 or Slc23a2 are unaltered compared with wild-type mice, this would support the authors' conclusion that dysfunction of NKA alpha2 or alpha3 is responsible for the low ascorbic acid levels.

7. In the Discussion, it is stated that seizures are exaggerated by low ASC in a mouse model. The authors should elaborate by stating which mouse model and human disease modelled they are referring to.

8. In the Discussion, it would be helpful if the authors would explain how they might, in future, test their hypothesis that "decreased levels of ASC relate to ATP1A2- and ATP1A3-related neurologic disorders".

9. The title of the manuscript (Decreased Content of Ascorbic acid (Vitamin C) in the Brain of Mouse Models of Na+, K+-ATPase-related Neurologic Disorders) does not state that the subjects of the study are actually NKA alpha2 and NKA alpha3 KO mice. A more informative title would be: 'Decreased Content of Ascorbic acid (Vitamin C) in the Brain of Knockout Mouse Models of Na+, K+-ATPase-related Neurologic Disorders'. This is important because the vast majority of NKA-related disorders are caused by missense mutations rather than deletions or nonsense mutations of ATP1A2 and ATP1A3.

10. The Introduction refers to 'a small number of AHCs'. This should be rephrased to clarify what this means.

11. There are a few typographical issues:

- 'Na+, K+-ATPase' is more commonly written without a space between the , and K: Na+,K+-ATPase.

- 'the homozygous knockout neonatal brain (Atp1a3-/-) (Table 1) [32,43],.' - delete the , after ].

- 'The ASC content in the cortex and basal ganglia also decreased in the peripartum α3-HT mice' - change to 'The ASC content in the cortex and basal ganglia was also decreased in the peripartum of α3-HT mice'.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-20-29560R1

Decreased content of ascorbic acid (vitamin C) in the brain of knockout mouse models of Na+,K+-ATPase-related neurologic disorders

PLOS ONE

Dear Dr. Ikeda,

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 submit your revised manuscript by Mar 06 2021 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Yuqing Li, Ph.D.

Academic Editor

PLOS ONE

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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: (No Response)

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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

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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 #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

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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 #2: All of my comments have been adequately addressed apart from my point on the ambiguity of the following passage: "Surviving 1-cell embryos were transferred into the oviducts of pseudopregnant ICR females. Slc23a2-KO (strain deposited in the RIKEN BioResource Research Center) and Slc23a2-floxed mice were generated as previously described [40,41]. Because all the latter were found to be random integrations in non-homologous chromosomes, we used only Slc23a2-KO mice for the subsequent analyses." Please clarify in the manuscript what is being referred to by "all the latter".

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Reviewer #2: No

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Decreased content of ascorbic acid (vitamin C) in the brain of knockout mouse models of Na+,K+-ATPase-related neurologic disorders

PONE-D-20-29560R2

Dear Dr. Ikeda,

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.

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Kind regards,

Yuqing Li, Ph.D.

Academic Editor

PLOS ONE

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 #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: (No Response)

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

Reviewer #2: (No Response)

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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 #2: (No Response)

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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: (No Response)

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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 #2: (No Response)

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Reviewer #2: No