14-3-3 proteins promote synaptic localization of N-methyl d-aspartate receptors (NMDARs) in mouse hippocampal and cortical neurons

Gloria S. Lee; Jiajing Zhang; Yuying Wu; Yi Zhou

doi:10.1371/journal.pone.0261791

Peer Review History

Original SubmissionAugust 18, 2021
29 Sep 2021 Decision Letter - Jean-Pierre Mothet, Editor PONE-D-21-2681614-3-3 proteins promote synaptic localization of N-methyl d-aspartate receptors (NMDARs) in mouse hippocampal and cortical neuronsPLOS ONE Dear Dr. Zhou, 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. I have obtained two reviews which are included below. The comments are favorable but one reviewer raised some concerns that should be constructively addressed during revision. Notably, the reviewer is asking for controls regarding the specificity of dipofein action on NMDA receptors trafficking and whether the action is direct or undirect. Another major issue concerns the very faint or even absent signal for GluN1 in co-immunoprecipitation. It is indeed very difficult to detect the presence of GluN1. Therefore, confirming these results by using another experimental approach as proposed would be welcome. Please submit your revised manuscript by Nov 13 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. 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'. 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[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: Partly 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: The present manuscript investigates the role of 14-3-3 proteins in the modulation of synaptic NMDA receptor levels, focusing on GluN1, GluN2A and GluN2B subunits. In particular, the authors used two different in vitro models such as primary neurons and heterologous systems to understand how 14-3-3 affect receptor synaptic localization and forward trafficking. Even if results are potentially interesting and the effect shown in Fig.1-3 are relevant, experiments performed in heterologous cells are less convincing and should be reinforced with other data obtained using primary neurons. Here below main issues to be considered in the Results section. Figure 1-3 show very interesting results on the effect of 14-3-3 inhibition on synaptic NMDA receptors. However, these assays need key controls. Firstly, considering that blocks the ability of 14.3.3 to bind to target proteins such as Raf-1, the authors should provide evidence in their experimental setting of a direct effect of dipofein on the formation of NMDAR/14-3-3 complex or the existence of other indirect mechanisms. Moreover, the authors should also provide data about the specificity of the observed effect, i.e. analyse the effect of the inhibitor on synaptic levels of other receptors such AMPA or other postsynaptic membrane proteins. In addition, the first sentence of the Results section is not clear. Please reconsider this sentence and explain also in the Result section the activity of difopein. It will help for an easier understanding of the experimental approach. Results presented in Figure 4A-4B does not have a physiological relevance and their rationale remains unclear. It’s known that in neurons GluN1 forms early dimers with GluN2- or GluN3-type subunits. Accordingly, biotinylation experiments performed in hetelogous cells with single GluN1 transfection have limited value. Please explain or remove these assays. In addition, in all panels of Figure 4 representative images of GluN1 and GluN2B are of low quality, multiple bands for GluN2B and faint bands (I guess almost impossible to quantify) for GluN1 in the “S” samples. Please address these issues. Again, in Figure 6, the quality of the CO-IP bands does not allow for a proper support of the conclusions raised by the authors. In particular, the presence of GluN1 coprecipitation in Fig.6C is really very difficult to detect. The authors should confirm these results by using another experimental approach such as proximity ligation assay. Moreover, having these unclear data in heterologous cells overexpressing the two proteins opens the question about the presence and the amount of endogenous complex in neurons. Proximity ligation assay would a feasible approach also for this type of assays in neurons. Reviewer #2: This is an interesting article on the role of 14-3-3 proteins in NMDA receptor localisation, it is well writen and shows a strong case for 14-3-3 proteins being involved in NMDA receptor cell surface expression. The only minor comment I have for the paper is that Figure 4 has very small text size on panels B, D, E, G and H, which makes it fairly hard to read. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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Revision 1
16 Nov 2021 Author Response Response to Reviewers We thank the editor and reviewers for their enthusiasm and constructive comments and suggestions. The manuscript has been revised in response to reviewers’ comments. Our point-by-point responses are detailed below. Reviewer #1: Comment 1: “Figure 1-3 show very interesting results on the effect of 14-3-3 inhibition on synaptic NMDA receptors. However, these assays need key controls. Firstly, considering that blocks the ability of 14.3.3 to bind to target proteins such as Raf-1, the authors should provide evidence in their experimental setting of a direct effect of dipofein on the formation of NMDAR/14-3-3 complex or the existence of other indirect mechanisms” Response to Comment 1: Thank you for raising this important point. We agree that the difopein-related experiments alone are not sufficient to establish a direct link between 14-3-3/NMDAR complex and synaptic targeting of NMDARs. In order to address this question, we created mutations on two putative 14-3-3 protein binding sites and tested their interaction with GluN2A and GluN2B subunits. However, those mutations did not significantly reduce 14-3-3 binding to these subunits (S2 Fig). It poses difficulty for us to generate 14-3-3 binding-deficient NMDAR mutants to directly assess the contribution of 14-3-3/NMDAR complex in synaptic targeting of these receptors. To clarify this issue, we added the following sentences in the revised manuscript (“Discussion section”: line 539): “Considering that difopein can inhibit 14-3-3 binding to a number of proteins in neurons, 14-3-3 proteins may indirectly affect synaptic localization and surface expression of NMDARs through their interactions with other proteins. Thus, future studies are needed to further determine the molecular details of the 14-3-3/NMDAR complex. This may help generate 14-3-3 binding-deficient NMDAR mutants to assess the role of 14-3-3/NMDAR interaction in synaptic targeting of these receptors.” Comment 2: “Moreover, the authors should also provide data about the specificity of the observed effect, i.e. analyse the effect of the inhibitor on synaptic levels of other receptors such AMPA or other postsynaptic membrane proteins.” Response to Comment 2: Thank you for this question. Previously, we have done experiments to investigate the specificity of the effect of difopein on postsynaptic membrane proteins in vivo using the 14-3-3 FKO mice, which expresses difopein in the forebrain region (also referred to as the 132-founder line). The 14-3-3 FKO mice exhibited lowered levels of synaptic NMDARs (GluN1 and GluN2A subunits) in hippocampal PSD fractions, whereas other postsynaptic membrane proteins including AMPARs (GluA1, GluA2), kainite receptors (GluK2/3), and PSD-95 were not affected (Fig 6, Qiao et al., 2014). In addition, 14-3-3 FKO mice exhibited reduction of NMDA/AMPA ratio without changes in fractions of currents mediated by AMPARs in CA1 hippocampal neurons (Fig 5A). These mice also exhibited lowered NMDAR-mediated EPSCs in CA1 hippocampal neurons (Fig 5B). For your reference, we attached published Figure 5 and Figure 6 below (Qiao, Foote, Graham, Wu, & Zhou, 2014). Comment 3: “In addition, the first sentence of the Results section is not clear. Please reconsider this sentence and explain also in the Result section the activity of difopein. It will help for an easier understanding of the experimental approach” Response to Comment 3: We appreciate your suggestion. We rewrote the first sentence from the Results section to make this sentence clearer. Below is the revised sentence (“Results section”: line 290): “To investigate the role of 14-3-3 proteins in synaptic localization of NMDARs, we assessed the effect of 14-3-3 inhibition on synaptic levels of NMDARs in primary glutamatergic cortical and hippocampal neurons. In this experiment, neurons were infected with the Ca2+/calmodulin-dependent protein kinase II α (CAMKIIα) promoter-driven adeno-associated virus (AAV) that expresses either YFP-fused difopein or YFP (control). Difopein is an established 14-3-3 inhibitor that binds to 14-3-3 proteins with high affinity, thereby competitively inhibits 14-3-3 ligand interactions (Masters & Fu, 2001).” Comment 4: “Results presented in Figure 4A-4B does not have a physiological relevance and their rationale remains unclear. It’s known that in neurons GluN1 forms early dimers with GluN2- or GluN3-type subunits. Accordingly, biotinylation experiments performed in hetelogous cells with single GluN1 transfection have limited value. Please explain or remove these assays.” Response to Comment 4: Thank you for this suggestion. We agree that results presented in Figure 4A-4B do not have a physiological relevance, so we removed Figure 4A-4B in the revised manuscript. Comment 5: “In addition, in all panels of Figure 4 representative images of GluN1 and GluN2B are of low quality, multiple bands for GluN2B and faint bands (I guess almost impossible to quantify) for GluN1 in the “S” samples. Please address these issues.” Response to Comment 5: We thank you for your observation. It is quite challenging to transiently express NMDAR subunits in heterologous system and to achieve sufficient level of surface NMDARs for the following reasons: 1) fast turnover rate of the NMDAR subunit proteins in these cells, 2) nonspecificity of the commercially available antibodies, and 3) sensitivity of the antigen-antibody interaction to denaturing conditions. However, to ensure that we properly quantify bands corresponding to the NMDAR subunits, we not only identified those bands based on their molecular weights and compared to the sizes of endogenous NMDAR subunits in mouse brain lysates, but also used non-transfected tsA201 cell lysates as a negative control. We are also quite confident in our quantification method for this assay, as we used the same setting to scan our blots and believe that our quantified data is a better measure than the representative blot image shown in this figure. Comment 6: “Again, in Figure 6, the quality of the CO-IP bands does not allow for a proper support of the conclusions raised by the authors. In particular, the presence of GluN1 coprecipitation in Fig.6C is really very difficult to detect. The authors should confirm these results by using another experimental approach such as proximity ligation assay. Moreover, having these unclear data in heterologous cells overexpressing the two proteins opens the question about the presence and the amount of endogenous complex in neurons. Proximity ligation assay would a feasible approach also for this type of assays in neurons.” Response to Comment 6: Thank you for your comment. We have a conducted new experiment demonstrating that 14-3-3 protein can clearly coIP with GluN1, GluN2A, and GluN2B subunits from hippocampal brain lysates of wild type mice (S1 Fig). This result provides support for the existence of the endogenous NMDAR/14-3-3 complex in the brain. However, we do realize that our coIP results still do not conclude that 14-3-3 directly binds to NMDARs. The proximity ligation assay will be a more advanced way to directly address the biochemical nature of this protein complex. We thank you for the suggestion and plan to adopt this assay in our future experiments. Reviewer #2: Comment 1: “This is an interesting article on the role of 14-3-3 proteins in NMDA receptor localisation, it is well written and shows a strong case for 14-3-3 proteins being involved in NMDA receptor cell surface expression. The only minor comment I have for the paper is that Figure 4 has very small text size on panels B, D, E, G and H, which makes it fairly hard to read.” Response to Comment 1: Thank you for your comments. As suggested, we changed the text size on panels B, C, E, and F (previously D, E, G, and H) for Figure 4. As suggested by reviewer 1, we also removed Figure 4A and B in the revised manuscript. References Masters, S. C., & Fu, H. (2001). 14-3-3 proteins mediate an essential anti-apoptotic signal. J Biol Chem, 276(48), 45193-45200. doi:10.1074/jbc.M105971200 Qiao, H., Foote, M., Graham, K., Wu, Y., & Zhou, Y. (2014). 14-3-3 proteins are required for hippocampal long-term potentiation and associative learning and memory. J Neurosci, 34(14), 4801-4808. doi:10.1523/JNEUROSCI.4393-13.2014 Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0261791.r002
10 Dec 2021 Decision Letter - Jean-Pierre Mothet, Editor 14-3-3 proteins promote synaptic localization of N-methyl d-aspartate receptors (NMDARs) in mouse hippocampal and cortical neurons PONE-D-21-26816R1 Dear Dr. Zhou, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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 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, Jean-Pierre Mothet, 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 #1: All comments have been addressed Reviewer #2: All comments have been addressed ******** 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: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: (No Response) Reviewer #2: Yes ****** 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: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: (No Response) Reviewer #2: The authors have addresses the points I have previously made, by changing the size of the font in figure 4. ****** 7. 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 https://doi.org/10.1371/journal.pone.0261791.r003
Formally Accepted
16 Dec 2021 Acceptance Letter - Jean-Pierre Mothet, Editor PONE-D-21-26816R1 14-3-3 proteins promote synaptic localization of N-methyl d-aspartate receptors (NMDARs) in mouse hippocampal and cortical neurons Dear Dr. Zhou: I'm 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 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 plosone@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 Jean-Pierre Mothet Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0261791.r004

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