Copper supplementation enhances pigmentation and induces dopamine production in ARPE19

Hironori Uehara; Baila Shakaib; Sangeetha Ravi Kumar; Bonnie Archer; Balamurali Ambati

doi:10.1371/journal.pone.0327352

Peer Review History

Original SubmissionFebruary 10, 2025
Decision Letter - Subbulakshmi Chidambaram, Editor PONE-D-25-05806Copper supplementation enhances pigmentation and induces dopamine production in ARPE19PLOS ONE Dear Dr. Uehara, 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. The reviewers' major concerns pertain to the integrity of the cells and the quantitative measurement of pigmentation. Please provide additional evidence and clarification regarding the viability and structural integrity of the cells used in your experiments. Further, the methodology for measuring and analyzing pigmentation requires more rigorous quantitative approaches and appropriate statistical validation. Please thoroughly address these concerns along with all other reviewer queries in your revision. Please submit your revised manuscript by May 01 2025 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. 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Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. [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: Yes Reviewer #2: Yes Reviewer #3: No ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: Yes Reviewer #3: No ****** 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 Reviewer #3: 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 Reviewer #3: 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: In this article, the authors investigates the effect of copper supplementation on dopamine biosynthesis in ARPE19 cells, proposing a pathway in which tyrosinase mediates dopamine biosynthesis in RPE cells. The results are interesting, particularly in reporting copper-induced dopamine synthesis in non-neuronal cells. However, the manuscript requires further refinement in mechanistic exploration and experimental rigor. Below are specific comments: 1. The image quality of Figures 1C and E is insufficient to clearly discern changes in cellular pigmentation. Additionally, quantitative analysis of melanin should be included. 2. Quantitative analysis for Tyrosinase protein expression, showed in Figure 4C, should be included to clarify its role in dopamine synthesis. 3. Include cell viability assays (e.g., MTT or CCK-8) to rule out potential cytotoxicity from copper supplementation. 4. It is better to provide additional mechanistic evidence for tyrosinase protein stabilization, such as protein degradation assays (e.g., CHX chase) to confirm copper's role. 5. Provide more details on sample handling for dopamine ELISA, particularly measures to prevent dopamine degradation. 6. More background on the rationale for using ARPE19 cells as a model, especially in comparison to other RPE models (e.g., iPSC-derived RPE), would strengthen the introduction. 7. The discussion on "non-neuronal dopamine biosynthesis" could be expanded, particularly regarding existing research on dopamine sources in peripheral tissues. 8. Further explore the reasons for the lack of dopamine synthesis with short-term copper supplementation (Figure 3B), such as copper transport efficiency or intracellular copper dynamics. 9. Provide more literature support for the biological functions of RPE-derived dopamine, especially its mechanisms in choroidal thickness and myopia development. 10. Define abbreviations (e.g., DDC, L-DOPA) upon first use. Reviewer #2: In this manuscript Uehara and coworkers test the hypothesis that tyrosinase-derived L-DOPA could be converted to dopamine and show that supplementation of ARPE19 medium with copper putatively increases pigmentation. The study tests an interesting hypothesis that could be of significant importance. The authors do show an increase in dopamine production using ELISA which is supportive of the proposed hypothesis, however the study as performed is incomplete for the following reasons. First the authors do little to quantify and characterize the putative increase in pigment induced by copper supplementation. They depend on qualitative visual observations and frankly the quality of figure 2 is less than compelling. Second, While they show an increase in dopamine expression by ELISA, they focus on tyrosinase expression and ignore expression of aromatic L-amino acid decarboxylase (AADC), the enzyme that catalyzes the conversion of L-dopa to dopamine. The authors could significantly improve the manuscript by addition of the following: 1. Melanin pigment should be quantitatively assessed. One approach might be to use a spectrophotometric assay like that of Hu (PMID: 18435617). 2. Figure 2 is not terribly compelling as a visual representation of pigmentation. Better quality photos are required and should include photomicrographs at a cellular level showing pigment granules. 3. The authors should examine expression of AADC. This should include assay for AADC mRNA and/or protein as well as assay of AADC activity in cell lysates both with and without Copper supplementation. Reviewer #3: The authors have conducted a study investigating enhanced pigmentation and dopamine production after copper supplementation in ARPE19 cells. The outline of the study is well understood, however there are some shortcomings that lead me to suggest that the study not be accepted. - Statistical accounting is missing overall. This is a serious flaw. - ARPE19 cells have high turnover and usually used for many cycles. They can easily undergo a "drift" over time, which can express itself genetically, morphologically, in growth patterns, etc. The authors need to at least include check points to control for the drift. - Pigmentation has been assessed visually, but an objective measurement method needs to be introduced. ****** 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 Reviewer #3: 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.0327352.r001
Revision 1
29 Apr 2025 Author Response Reviewer #1: In this article, the authors investigates the effect of copper supplementation on dopamine biosynthesis in ARPE19 cells, proposing a pathway in which Tyrosinase mediates dopamine biosynthesis in RPE cells. The results are interesting, particularly in reporting copper-induced dopamine synthesis in non-neuronal cells. However, the manuscript requires further refinement in mechanistic exploration and experimental rigor. Below are specific comments: 1. The image quality of Figures 1C and E is insufficient to clearly discern changes in cellular pigmentation. Additionally, quantitative analysis of melanin should be included. We added higher magnification images (Supplemental Figure 4) and melanin quantification (Supplemental Figure 5). 2. Quantitative analysis for Tyrosinase protein expression, showed in Figure 4C, should be included to clarify its role in dopamine synthesis. We quantified the tyrosinase band (Figure 4D). Also, we clarified tyrosinase activity (tyrosine to L-DOPA) in the result section. 3. Include cell viability assays (e.g., MTT or CCK-8) to rule out potential cytotoxicity from copper supplementation. We conducted a CCK-8 assay for a short-term culture (1-4 days) and crystal violet for a mid-term culture (1-14 days). Since our ARPE19 culture for maturation would require over 90-100 days, the protein concentrations of the 105-day culture were evaluated for potential copper toxicity. In our conditions, we did not find significant toxicity; copper supplements (10uM) indicate that cell survival increased slightly. However, this result does not guarantee the increase of cell survival since copper also impacts the extracellular matrix (e.g., collagen cross-linking enzymes such as lysyl oxidase). Therefore, we concluded, "Copper did not show significant cell toxicity in our condition." These results are shown in Supplemental Figures 1-3. 4. It is better to provide additional mechanistic evidence for tyrosinase protein stabilization, such as protein degradation assays (e.g., CHX chase) to confirm copper's role. We agree with the reviewer's point. However, there are several challenges in addressing this: A) The long-term culture is necessary. We also recognize that this resulted in more deviations in tyrosinase expression between the samples. B) Non-copper culture medium could contain residual copper from 1%FBS. Therefore, tyrosinase in non-copper-treated cells could bind copper from the medium. C) Since copper is an indispensable element for cell survival, such as cytochrome c oxidase in mitochondria, the complete chelation of copper may cause malfunction of the cells. D) Most copper binds to serum proteins such as albumin and ceruloplasmin. Removing the copper from the protein may require a special type of dialysis, such as albumin dialysis. Therefore, instead of CHX experiments, the discussion about tyrosinase stability was expanded as below. " Tyrosinase requires multiple steps for maturation in the ER (endoplasmic reticulum) and Golgi, with strict quality controls. Although copper is not indispensable for tyrosinase processing/translocation, the holo-enzyme of tyrosinase showed higher stability than apo-enzyme 5. Provide more details on sample handling for dopamine ELISA, particularly measures to prevent dopamine degradation. About the sample handling, we added metabisulfite (final conc. 4mM) and EDTA (final conc. 1mM). Also, the samples were kept on ice until the experiment, and we started the ELISA within 30 minutes after harvesting the culture medium. We added these points to the method. This ELISA kit contains unique methods to prevent dopamine: cis-diol affinity extraction, acylation, and enzyme conversion. This process prevents dopamine degradation. We also asked the manufacturer for references regarding dopamine degradation; unfortunately, they do not have a paper to show the above, but they agreed with the above statement. Also, they said, "the dopamine would be most suspectable to degradation (pH> 7,5). However, the buffer added for extraction contains EDTA. Also important is that the standards undergo the same procedure, and if degradation occurs, this will also be the case for the standards." 6. More background on the rationale for using ARPE19 cells as a model, especially in comparison to other RPE models (e.g., iPSC-derived RPE), would strengthen the introduction. Yes, we added more background about ARPE19 and its culture cost are much more affordable compared to the iPSC-derived RPE. Also, the provider of the iPSC-derived RPE does not disclose the composition of their culture medium. Application of the current culture condition to iPSC-derived RPE may cause unexpected results. Therefore, we used APRE19 in this study (copper effect on tyrosinase and dopamine). However, we will use iPSC-derived RPE for future experiments. 7. The discussion on "non-neuronal dopamine biosynthesis" could be expanded, particularly regarding existing research on dopamine sources in peripheral tissues. In the introduction, we expanded the discussion of non-neuronal dopamine biosynthesis. 8. Further explore the reasons for the lack of dopamine synthesis with short-term copper supplementation (Figure 3B), such as copper transport efficiency or intracellular copper dynamics. We expanded the discussion about copper transport. 9. Provide more literature support for the biological functions of RPE-derived dopamine, especially its mechanisms in choroidal thickness and myopia development. We added additional references. However, most literature studied retinal dopamine (neuronal retina) or ocular dopamine (whole eye). Therefore., in the introduction, we discussed dopamine's general biological function, not specifically that of of RPE-derived dopamine. 10. Define abbreviations (e.g., DDC, L-DOPA) upon first use. We apologize for the earlier oversight. We have now defined the abbreviations in the first appearance. Reviewer #2: In this manuscript Uehara and coworkers test the hypothesis that tyrosinase-derived L-DOPA could be converted to dopamine and show that supplementation of ARPE19 medium with copper putatively increases pigmentation. The study tests an interesting hypothesis that could be of significant importance. The authors do show an increase in dopamine production using ELISA which is supportive of the proposed hypothesis, however the study as performed is incomplete for the following reasons. First the authors do little to quantify and characterize the putative increase in pigment induced by copper supplementation. They depend on qualitative visual observations and frankly the quality of figure 2 is less than compelling. Second, While they show an increase in dopamine expression by ELISA, they focus on tyrosinase expression and ignore expression of aromatic L-amino acid decarboxylase (AADC), the enzyme that catalyzes the conversion of L-dopa to dopamine. The authors could significantly improve the manuscript by addition of the following: 1. Melanin pigment should be quantitatively assessed. One approach might be to use a spectrophotometric assay like that of Hu (PMID: 18435617). We added the melanin quantification result as Supplemental Figure 5. 2. Figure 2 is not terribly compelling as a visual representation of pigmentation. Better quality photos are required and should include photomicrographs at a cellular level showing pigment granules. We added high-magnification images as Supplemental Figure 4. 3. The authors should examine expression of AADC. This should include assay for AADC mRNA and/or protein as well as assay of AADC activity in cell lysates both with and without Copper supplementation. We examined DDC mRNA expression (Figure 4) as the equivalent of AADC expression. AADC is a more common name than DDC, but we followed HGNC (HUGO Gene Nomenclature Committee), which uses DDC as the approved name. To reduce the confusion, we stated this more clearly at the first appearance of DDC. Reviewer #3: The authors have conducted a study investigating enhanced pigmentation and dopamine production after copper supplementation in ARPE19 cells. The outline of the study is well understood, however there are some shortcomings that lead me to suggest that the study not be accepted. 1. Statistical accounting is missing overall. This is a serious flaw. We apologize for the earlier oversight, and we have now added a statistical section to the methods. 2. ARPE19 cells have high turnover and usually used for many cycles. They can easily undergo a "drift" over time, which can express itself genetically, morphologically, in growth patterns, etc. The authors need to at least include check points to control for the drift. Thank you for your valuable feedback regarding genetic drift in our cell culture experiments. We agree that genetic drift can significantly impact genotype, phenotype, and overall cell functionality. Subculturing and increasing passage numbers are recognized as primary contributors to this drift. To minimize such effects, we prepared cryo-stocks of ARPE19 cells in passage 2 upon receipt from ATCC and used them for experiments in passages 4–5. Also, the cells were allowed to mature in 1% FBS without subculture. This approach should mitigate genetic drift, and we believe its impact in our experiments was minimal to moderate. While cryopreservation is a common strategy to limit genetic drift and loss of cell characteristics, we were unable to perform confirmatory genomic analyses due to limited resources. We will consider including genetic drift assessments in future studies. We appreciate your understanding and the opportunity to address this point. 3. Pigmentation has been assessed visually, but an objective measurement method needs to be introduced. We quantified the melanin in the cell lysate and showed it in Supplemental Figure 5. Attachments Attachment Submitted filename: CopperdopamineResponse-Point-by-Point20250428.docx https://doi.org/10.1371/journal.pone.0327352.r002
Decision Letter - Subbulakshmi Chidambaram, Editor PONE-D-25-05806R1Copper supplementation enhances pigmentation and induces dopamine production in ARPE19PLOS ONE Dear Dr. Uehara, 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. The changes made in response to the previous comments are satisfactory. However, we noted that some supplementary figures, especially, Suppl Fig.4 and 5 are important for the proper interpretation of your findings. Therefore, to ensure clarity and full accessibility of the key data to the readers, I kindly request you to place those images in the main manuscript and update the figure numbering accordingly. Please submit your revised manuscript by Jul 20 2025 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, Subbulakshmi Chidambaram, Ph.D Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. Additional Editor Comments : The changes made in response to the previous comments are satisfactory. However, we noted that some supplementary figures, especially, Suppl Fig.4 and 5 are important for the proper interpretation of your findings. Therefore, to ensure clarity and full accessibility of the key data to the readers, I kindly request you to place those images in the main manuscript and update the figure numbering accordingly. [Note: HTML markup is below. Please do not edit.] [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.0327352.r003
Revision 2
6 Jun 2025 Author Response We appreciate the opportunity to submit the revised version of our manuscript. In response to the latest comment, we have revised the manuscript as follows: 1) S4 Fig and S5 Fig have been moved to Fig 3 and Fig 4, respectively. 2) Figure numbers and legends have been adjusted accordingly throughout the manuscript. Please let us know if any further modifications are required. Thank you again for your time and consideration. https://doi.org/10.1371/journal.pone.0327352.r004
Decision Letter - Subbulakshmi Chidambaram, Editor Copper supplementation enhances pigmentation and induces dopamine production in ARPE19 PONE-D-25-05806R2 Dear Dr. Uehara, 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, Subbulakshmi Chidambaram, Ph.D Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0327352.r005
Formally Accepted
Acceptance Letter - Subbulakshmi Chidambaram, Editor PONE-D-25-05806R2 PLOS ONE Dear Dr. Uehara, 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 You will receive further instructions from the production team, including instructions on how to review your proof when it is ready. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few days 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. Subbulakshmi Chidambaram Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0327352.r006

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