Pharmacological inhibition of carnitine palmitoyl transferase 1 inhibits and reverses experimental autoimmune encephalitis in rodents

Anne Skøttrup Mørkholt; Michal Krystian Oklinski; Agnete Larsen; Robert Bockermann; Shohreh Issazadeh-Navikas; Jette Goller Kloth Nieland; Tae-Hwan Kwon; Angelique Corthals; Søren Nielsen; John Dirk Vestergaard Nieland

doi:10.1371/journal.pone.0234493

Peer Review History

Original SubmissionDecember 23, 2019
25 Mar 2020 Decision Letter - Fulvio D'Acquisto, Editor PONE-D-19-35522 Pharmacological inhibition of carnitine palmitoyl transferase 1 inhibits and reverses experimental autoimmune encephalitis in rodents PLOS ONE Dear Mr Nieland, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. We would appreciate receiving your revised manuscript by May 09 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Fulvio D'Acquisto, PhD Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Thank you for stating the following in the Financial Disclosure section: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." We note that one or more of the authors are employed by a commercial company: "Meta-IQ, ApS" a) Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form. Please also include the following statement within your amended Funding Statement. “The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.” If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement. b) Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc. Within your Competing Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. Please respond by return email with an updated Funding Statement and Competing Interests Statement and we will change the online submission form on your behalf. Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests Additional Editor Comments (if provided): Please try to address as many reasonable comments as possible. [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: No Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Authors of this study have demonstrated that etomoxir effectively mitigates the loss of myelin and other pro-inflammatory, degenerative processes in brain tissue of mouse and rat models of experimental autoimmune encephalitis (EAE), which recapitulates many of the same pathological characteristics as MS in humans. Despite this interesting finding, authors show only superficially descriptive data here and there is no mechanistic insights provided as to why this might be happening. They make the argument that inhibition of CPT1A by etomoxir is a primary reason for the therapeutic effect, yet there are no convincing measurements taken here to support that. Specific areas where this study would be strengthened are as follows: 1) Although etomoxir is indeed an irreversible inhibitor of CPT1A, it also has many off-target effects on other lipid metabolism pathways, including activation of PPARs, and some effects on CoA metabolism. These are only some of the known off-target effects of this molecule, there likely are many more. Thus, many experiments are needed to confirm that CPT1A inhibition is the primary MoA for etomoxir in a study such as this. These experiments are not sufficiently performed in this study. Specifically, no measurements of fatty acid oxidation are performed in any cell or tissue from the EAE models to determine if etomoxir is having an effect on this pathway. The immunostaining for CPT1A protein in the brain slices shown here by the authors is essentially meaningless, as this could be driven by multiple players, including the disease pathology. The functional effect of etomoxir on blunting fatty acid oxidation is not confirmed. 2) It is not at all clear, given the experimental design here, which cell type is relevant to the etomoxir effect. Is CPT1A inhibition more important in oligodendrocytes, lymphocytes, macrophages, or all of them? This is not at all clear from this study. Work in cell culture models might be useful to sort this out, or single-cell experiments from the EAE models. 3) Given the numerous off-target effects of etomoxir, an additional experiment that should be considered is to use a targeted CPT1A-deficient mouse line to confirm that CPT1A is mechanistically involved in the progression of EAE. This would greatly strengthen the etomoxir data. 4) Use of ferritin staining as a marker of ‘mitochondrial dysfunction’ is dubious, at best. If fatty acid oxidation is pathologically involved in EAE, then having some indication of mitochondrial fatty acid oxidation in the tissues involved in this process is essential here. It is well-known that mitochondrial fatty acid oxidation causes more oxidative stress, and in turn this could lead to degeneration of oligodendrocytes and inflammation, but these lines of investigation have not been followed here, and the use of ferritin as a marker does not address these questions in any meaningful way. 5) More discussion about EAE as a model of MS in humans is much needed here. What are the similarities, and what are the limitations? Reviewer #2: The article entitled,” Pharmacological Inhibition of carnitine palmitoyltransferase 1 inhibits and reverses experimental autoimmune encephalitis in rodents” by Morkholt et al., describes the mechanism of action of a CPT1 inhibitor in vivo. Overall, experiments performed and data analysis are appropriate, and the manuscript can be published in PLOS One after the authors provide some clarification. 1. Fig 3a and b. The authors indicate that rats that received CPT1 inhibitor etomoxir showed lower clinical scores compared to placebo. However, the results were significant only for day 11 data. Furthermore, there was a significant change in body weight. In fact, compared to the change in body weight, the clinical score was less significant. The authors need to explain this. 2. The respiratory chain is an important aspect of cellular metabolism. Inhibition of CPT1 is affected in all cells in the body. Is there any selectivity with CPT1 inhibitors only to neuronal cells? ****** 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: 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 to be viewed.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0234493.r001
Revision 1
11 May 2020 Author Response PLOS ONE Decision: Revision required PONE-D-19-35522 Pharmacological inhibition of carnitine palmitoyl transferase 1 inhibits and reverses experimental autoimmune encephalitis in rodents Reviewer #1: Authors of this study have demonstrated that etomoxir effectively mitigates the loss of myelin and other pro-inflammatory, degenerative processes in brain tissue of mouse and rat models of experimental autoimmune encephalitis (EAE), which recapitulates many of the same pathological characteristics as MS in humans. Despite this interesting finding, authors show only superficially descriptive data here and there is no mechanistic insights provided as to why this might be happening. They make the argument that inhibition of CPT1A by etomoxir is a primary reason for the therapeutic effect, yet there are no convincing measurements taken here to support that. Specific areas where this study would be strengthened are as follows: 1) Although etomoxir is indeed an irreversible inhibitor of CPT1A, it also has many off-target effects on other lipid metabolism pathways, including activation of PPARs, and some effects on CoA metabolism. These are only some of the known off-target effects of this molecule, there likely are many more. Thus, many experiments are needed to confirm that CPT1A inhibition is the primary MoA for etomoxir in a study such as this. These experiments are not sufficiently performed in this study. Specifically, no measurements of fatty acid oxidation are performed in any cell or tissue from the EAE models to determine if etomoxir is having an effect on this pathway. The immunostaining for CPT1A protein in the brain slices shown here by the authors is essentially meaningless, as this could be driven by multiple players, including the disease pathology. The functional effect of etomoxir on blunting fatty acid oxidation is not confirmed. Response: Several studies have shown that etomoxir increases the glucose metabolism and decreases the lipid metabolism in both mice and humans[1–5]. Furthermore, we have measured the glucose levels in serum from EAE mice, which is illustrated in the figure below. Cpt1a EAE (n=4), WT EAE placebo (n=3) and WT EAE etomoxir (n=3). 2) It is not at all clear, given the experimental design here, which cell type is relevant to the etomoxir effect. Is CPT1A inhibition more important in oligodendrocytes, lymphocytes, macrophages, or all of them? This is not at all clear from this study. Work in cell culture models might be useful to sort this out, or single-cell experiments from the EAE models. Response: CPT1a is expressed in all cells (except in muscle and adipose tissue). We and others have shown in the past that lymphocytes, macrophages dendritic cells etc. are all affected. In addition, astrocytes have shown to have upregulated CPT1a. The theory is that most cells are affected by the drug (etomoxir) and therefore, change the balance in the body to a more glucose metabolism, resulting in the clinical effect on the disease. 3) Given the numerous off-target effects of etomoxir, an additional experiment that should be considered is to use a targeted CPT1A-deficient mouse line to confirm that CPT1A is mechanistically involved in the progression of EAE. This would greatly strengthen the etomoxir data. Response: These data have in the meantime been published in Scientific Reports where it has been shown that CPT1a knock down mice are resistant to EAE development[6]. We have now referred to this study in section “Discussion” p. 17 l. 361-363. 4) Use of ferritin staining as a marker of ‘mitochondrial dysfunction’ is dubious, at best. If fatty acid oxidation is pathologically involved in EAE, then having some indication of mitochondrial fatty acid oxidation in the tissues involved in this process is essential here. It is well-known that mitochondrial fatty acid oxidation causes more oxidative stress, and in turn this could lead to degeneration of oligodendrocytes and inflammation, but these lines of investigation have not been followed here, and the use of ferritin as a marker does not address these questions in any meaningful way. Response: Dysfunctional mitochondria and decreased brain metabolism can promote an environment for enhanced oxidative stress. In the manuscript published in meantime, we have demonstrated that EAE severity correlates with the gene expression of proteins involved in oxidative stress such as HO- 1 and NOX2[6]. The results showed that the expression of NOX2 and HO-1was significantly increased in the WT EAE animals. These results were in accordance to the expression of MBP indicating degradation of myelin in the EAE animals. Ferritin, the iron storage protein, can when reaching excessive levels cause ROS. The levels of ferritin are found to be increased in MS patients compared to normal controls as an attempt to protect against the free levels/excess of iron[7]. We have now made changes to this comment as well as referred to the study in section “Discussion” p. 19 l. 404 and l. 406-409. 5) More discussion about EAE as a model of MS in humans is much needed here. What are the similarities, and what are the limitations? Response: The experimental autoimmune encephalomyelitis model is an in vivo state-of-art animal model of MS as it mimics the pathological and clinical features present in the disease. We have included more discussion of the use of EAE as a model for MS in section “Discussion” p. 20 l. 429-435. Reviewer #2: The article entitled,” Pharmacological Inhibition of carnitine palmitoyltransferase 1 inhibits and reverses experimental autoimmune encephalitis in rodents” by Morkholt et al., describes the mechanism of action of a CPT1 inhibitor in vivo. Overall, experiments performed and data analysis are appropriate, and the manuscript can be published in PLOS One after the authors provide some clarification. 1) Fig 3a and b. The authors indicate that rats that received CPT1 inhibitor etomoxir showed lower clinical scores compared to placebo. However, the results were significant only for day 11 data. Furthermore, there was a significant change in body weight. In fact, compared to the change in body weight, the clinical score was less significant. The authors need to explain this. Response: This is a good observation and comment of the reviewer. In this rat model the development of EAE was highly aggressive, and all placebo animals died within these 11 days. The effect of the disease induction on the feeding behavior has been found to be preceding the clinical score. In this model weight change is a first indication of disease induction, which therefore has a higher significance. 2) The respiratory chain is an important aspect of cellular metabolism. Inhibition of CPT1 is affected in all cells in the body. Is there any selectivity with CPT1 inhibitors only to neuronal cells? Response: Same answer as to Reviewer #1 question 2). CPT1a is expressed in all cells (except in muscle and adipose tissue). We and others have shown in the past that lymphocytes, macrophages dendritic cells etc. are all affected. In addition, astrocytes have shown to have upregulated CPT1a. The theory is that most cells are affected by the drug (etomoxir) and therefore, change the balance in the body to a more glucose metabolism, resulting in a reduced inflammation, reduced oxidative stress and the clinical effect on the disease. References 1. Timmers S, Nabben M, Bosma M, Bree B Van, Lenaers E, Beurden D Van. Augmenting muscle diacylglycerol and triacylglycerol content by blocking fatty acid oxidation does not impede insulin sensitivity. PNAS. 2012;109: 11711–11716. doi:10.1073/pnas.1206868109 2. Lopaschuk GD, Mcneil GF, Mcveigh JJ. Glucose oxidation is stimulated in reperfused ischemic hearts with the carnitine palmitoyltransferase 1 inhibitor , Etomoxir. 1989; 175–179. 3. Ratheiser K, Schneewiess B, Waldhausl W, Fasching P, Korn A, Nowotny P. Inhibition by Etomoxir of Carnitine Palmitoyltransferase I Reduces Hepatic Glucose Production and Plasma Lipids in Non-Insulin-Dependent Diabetes Mellitus. Metabolism. 1991;40: 1185–1190. 4. Shriver LP, Manchester M. Inhibition of fatty acid metabolism ameliorates disease activity in an animal model of multiple sclerosis. Sci Rep. 2011;1: 6–11. doi:10.1038/srep00079 5. Mørkholt AS, Wiborg O, Nieland JGK, Nielsen S, Nieland JD. Blocking of carnitine palmitoyl transferase 1 potently reduces stress-induced depression in rat highlighting a pivotal role of lipid metabolism. Sci Rep. 2017;7. doi:10.1038/s41598-017-02343-6 6. Mørkholt AS, Trabjerg MS, Krystian M, Oklinski E, Bolther L, Kroese LJ, et al. CPT1A plays a key role in the development and treatment of multiple sclerosis and experimental autoimmune encephalomyelitis. Sci Rep. 2019; 1–11. doi:10.1038/s41598-019-49868-6 7. Williams R, Buchheit C., Berman NE., Levine S. Pathogenic implications of iron accumulation in multiple sclerosis. J Neurochem. 2012;120: 7–25. doi:10.1111/j.1471- 4159.2011.07536.x.Pathogenic Attachments Attachment Submitted filename: Response to Reviewers.pdf https://doi.org/10.1371/journal.pone.0234493.r002
28 May 2020 Decision Letter - Fulvio D'Acquisto, Editor Pharmacological inhibition of carnitine palmitoyl transferase 1 inhibits and reverses experimental autoimmune encephalitis in rodents PONE-D-19-35522R1 Dear Dr. Nieland, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. With kind regards, Fulvio D'Acquisto, PhD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0234493.r003
Formally Accepted
1 Jun 2020 Acceptance Letter - Fulvio D'Acquisto, Editor PONE-D-19-35522R1 Pharmacological inhibition of carnitine palmitoyl transferase 1 inhibits and reverses experimental autoimmune encephalitis in rodents Dear Dr. Nieland: I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Professor Fulvio D'Acquisto Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0234493.r004

Open letter on the publication of peer review reports

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

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

Learn more at ASAPbio .