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Peer-Review Record

Expression of Melatonin and Dopamine D3 Receptor Heteromers in Eye Ciliary Body Epithelial Cells and Negative Correlation with Ocular Hypertension

Reviewer 1: Anonymous
Reviewer 2: Anonymous
Received: 20 November 2019 / Revised: 23 December 2019 / Accepted: 2 January 2020 / Published: 8 January 2020

Round 1

Reviewer 1 Report

The authors have found expression of melatonin and dopamine D3 receptor 2 heteromers in eye ciliary body epithelial cells and proved negative correlation with ocular hypertension.

 

Minor comments:

 

Line 38 In Abstract given statement “The underlying mechanisms, which impact on eye circadian rhythms, are not known” are not investigated in this study. Therefore, it is no clear why it is accented as a background of study in the Abstract. In Methods several sentences are not finished. For example, one of them: “Biophysical assays performed to identify interactions in heterologous systems”. It is no clear were assays performed or assays identified interactions? Overall in the article are misprints, like: Line 133 This work adhered adheres to guidelines elsewhere detailed. Line 522. “Dopamine, one of the main neurotransmitters of the central nervous system, participates in almost any higher function, from motor control to emotion control”. I am not sure about term “higher function”.

Major comment:

To my mind, no reason to use data obtained from two human glaucoma eye postmortem samples. I suggest to leave these data for the next article as it is impossible from 2 samples to get statistically significant results. Ethical Committee permission should be obtained if you use donor human eyes.

Author Response

 

Minor comments: 

Line 38 In Abstract given statement “The underlying mechanisms, which impact on eye circadian rhythms, are not known” are not investigated in this study. Therefore, it is no clear why it is accented as a background of study in the Abstract.

Answer: We understand the issue and, to address it, the “background” part of Abstract has been rewritten.

 

In Methods several sentences are not finished. For example, one of them: “Biophysical assays performed to identify interactions in heterologous systems”. It is no clear were assays performed or assays identified interactions?

Answer: We appreciate the comment that has led to careful reading of all sentences to, eventually, complete those that were not correct

 

 

Overall in the article are misprints, like: Line 133 This work adhered adheres to guidelines elsewhere detailed.

Answer: We appreciate the comment and we have made all effort to minimize/eliminate this kind of errors.

 

Line 522. “Dopamine, one of the main neurotransmitters of the central nervous system, participates in almost any higher function, from motor control to emotion control”. I am not sure about term “higher function”.

Answer: We appreciate the comment and we have replaced “higher” by “higher-order” as it is more correct. Both “higher” and “higher-order” are accepted in Neuroscience research to express human capabilities going from cognition to behaviour being, for instance “Cognition” composed of intellectual function, memory, speech and language, complex perception, orientation, attention, judgment, planning, and decision-making. We have now rewritten the sentence this way.

“Dopamine, one of the main neurotransmitters of the central nervous system, participates in almost any higher-order function, from motor control to emotion control and from cognition to behaviour” We think that it is not necessary to include the long “definition” unless otherwise stated by the reviewer.

 

Major comment:

To my mind, no reason to use data obtained from two human glaucoma eye postmortem samples. I suggest to leave these data for the next article as it is impossible from 2 samples to get statistically significant results. Ethical Committee permission should be obtained if you use donor human eyes. 

Answer. We are aware of the fact that n=2 is not enough to make statistical comparisons and this was already indicated in the summary:

from glaucoma cases (the trend was marked but no statistical analysis was possible as the number of available eyes was 2).

In what concern human eye results we have followed the instructions given in a previous paper (already accepted by Br J Pharmacol). Br J pharmacol reaised the same concern but considered that the data in human eyes was relevant and they wanted to have the data in the paper. In summary we think that it is quite relevant to keep the data from human eyes in the paper and to our understanding the data is presented in the right way (at least equal way that was acceptable for Br J Pharmacol). “Exploratory” was the work this journal suggested us and the one we used in the current paper (under consideration in Cells). In addition, we have not access in the short term to more human eyes. We notice that the ethical issues were not detailed in the first version and have been added.  Ethical issues are under tight control by the Spanish Government in any human tissue bank located in the Spanish territory, especially when there is the option (as in this case due to cornea transplantations) to use tissues to cure patients.

Reviewer 2 Report

The manuscript by Reyes-Resina and co-workers elaborates on the possible functional significance of the pineal hormone melatonin within the (patho-) physiological regulation of eye physiology. The authors use immortalized cell lines that were transfected to express both subtypes of the G-protein-coupled melatonin receptors , the MT1 and the MT2, together with various dopamine receptors. They observe a considerable heterodimerization of these receptors subtypes in vitro, which could, however, not been replicated in ex vivo human eye preparations. As there is some evidence for a link between melatonin in the mammalian retina and abnormal intraocular pressure, the authors conclude a possible therapeutic intervention strategy in glaucoma.

- Suprisingly, the well known coupling of MTs to a Gi was not confirmed in these studies.  This is irritating, as all studies to date point to a coupling of retinal MTs to an Gi-mediated inhibition of cAMP signaling.

- the authors use in some experiments a high pharmacological dose of melatonin (1 µM), which can by no means be interpreted as a physiological effect.

- to my knowledge there exists not one publication that shows a physiological importance of G-proetin coupled receptor dimerization. So far, it has only been described in several in vitro systems. The physiological consequence of hetereodimerization is so far unclear.

- the authors speculate in their 2 samples of human retinae about ‘a trend’. This is scientifically not adequate. Either, the authors have to increase sample numbers and are then able to conduct a statistical analysis on their results, or results should be omitted.

- the authors use MT-antibodies, distributed by ABCAM. To my knowledge, it has been notoriously difficult to generate valid MT antibodies. If ABCAM should have been successful, the authors have to validate these antibodies by, for example, showing immunohistochemical validation by comparison of eyes of wildtype and MT-knockout mice.

- it remains unclear, why the functional characterization of heteroceptors was moved into Appendix figures, as this is one major conclusion of importance of this study. Notably, these Appendix figures were not accessible to the reviewer.

 

MINOR COMMENTS

- page 2, line 88: these are not citations that link to the original cloning of the mammalian MTs.

- page 10, line 412: the authors should give a reference for the characterization of IIK7, which is here claimed to be a selective MT2 agonist.

- page 12, line 478: the authors should please explain, what is meant with ‘bi-directional cross-antagonism’.

- page 14, line 535: it is a vast over interpretation, to attribute the pineal gland to be ‘the most important gland in the mammalian body’. This has to be majorly weakened, regarding the adrenal, the pituitary the liver the gut as also important endocrine glands.

Author Response

Comments and Suggestions for Authors

The manuscript by Reyes-Resina and co-workers elaborates on the possible functional significance of the pineal hormone melatonin within the (patho-) physiological regulation of eye physiology. The authors use immortalized cell lines that were transfected to express both subtypes of the G-protein-coupled melatonin receptors , the MT1 and the MT2, together with various dopamine receptors. They observe a considerable heterodimerization of these receptors subtypes in vitro, which could, however, not been replicated in ex vivo human eye preparations. As there is some evidence for a link between melatonin in the mammalian retina and abnormal intraocular pressure, the authors conclude a possible therapeutic intervention strategy in glaucoma.

- Suprisingly, the well known coupling of MTs to a Gi was not confirmed in these studies.  This is irritating, as all studies to date point to a coupling of retinal MTs to an Gi-mediated inhibition of cAMP signaling.

Answer: Yes, indeed, this was quite surprising (very surprising) to us. Indeed the canonical coupling is to Gi; based on results on Gi-coupling in controls performed in cells individually expressing MT receptors, that were in figures in the Appendix on first submission, we preregistered a study, which has already been carried out, in which in the potency of melatonin and IIK7 in  HEK-293T cells expressing only one receptor was addressed. As the paper has been accepted and published prior to this one submitted to Cells, we have thought that it should be commented and referenced in the revised version of the paper.

- the authors use in some experiments a high pharmacological dose of melatonin (1 µM), which can by no means be interpreted as a physiological effect.

Answer: Again we have to mention the preregistered study. We were not aware that the affinity of melatonin (in binding studies) was very high. One of the aims of the preregistered study was to measure the potency to know why scientists, often use 1 microM melatonin for functional studies, despite the affinity is picomolar. Accordingly, we did what many scientist with much more experience than us in this field did: use 1 microM. Now we know that the potency is in the nanomolar range and that for one of the receptors it is not necessary to go so high but for the other it is convenient to go to 500 nM or higher. Finally we think that even today when we already know the differential potency in our hands, it would be still better to use the same concentration for MT1 and for MT2 receptors.

- to my knowledge there exists not one publication that shows a physiological importance of G-proetin coupled receptor dimerization. So far, it has only been described in several in vitro systems. The physiological consequence of hetereodimerization is so far unclear. 

Answer. We appreciate this opinion. This paper is an example of unexpected findings that can be explained by heteromer formation. In virtually all of our papers on heteromerization we provide data that, to the very least, suggest heteromer occurrence in natural sources. In addition we should indicate that the doubt in the field (e.g. after the last Symposia taking place in Stockholm last month) is with homomers but not with heteromers. But even when 200 rhodopsin molecules are placed randomly together, the system, using ab initio quantum mechanics approaches, evolves towards formation of stable homodimers. Heteromers characterized in our lab are also stable as deduced from both experimental and in silico approaches. Why some “couples” seem less stable using other sophisticated approaches deserves a discussion that is out of the scope of the present article. The number of interactions involving GPCRs is, as of today, >800 (many of them described in http://www.gpcr-hetnet.com/) and the majority of reporting laboratories do approach the issue also in physiological-like environments.

- the authors speculate in their 2 samples of human retinae about ‘a trend’. This is scientifically not adequate. Either, the authors have to increase sample numbers and are then able to conduct a statistical analysis on their results, or results should be omitted.

Comment to the Editor. This issue is raised the both reviewers. We will appreciate that after reading the query and the answers you as Cells’ Editor decide whether the data obtained in human eyes should be deleted or not. The decision by the handling Editor of Br. J. Pharmacol. in a similar situation in a recently accepted paper was to leave the data but indicating that these results were “exploratory” and (obviously) without attempting any statistical analysis. As we knew this, on submission to Cells we followed the advice on using “exploratory” and leave out any doubt concerning the number of eyes (n=2, already written in the abstract on first submission).

Answer. We provide the answer given to reviewer 1:

We are aware of the fact that n=2 is not enough to make statistical comparisons and this was already indicated in the summary:

from glaucoma cases (the trend was marked but no statistical analysis was possible as the number of available eyes was 2).

In what concern human eye results we have followed the instructions given in a previous paper (already accepted by Br J Pharmacol). Br J Pharmacol raised the same concern but considered that the data in human eyes was relevant and they wanted to have the data in the paper. In summary we think that it is quite relevant to keep the data from human eyes in the paper and to our understanding the data is presented in the right way (at least equal way that was acceptable for Br J Pharmacol). “Exploratory” was the work this journal suggested us and the one we used in the current paper (under consideration in Cells). In addition, we have not access in the short term to more human eyes. We notice that the ethical issues were not detailed in the first version and have been added.  Ethical issues are under tight control by the Spanish Government in any human tissue bank located in the Spanish territory, especially when there is the option (as in this case due to cornea transplantations) to use tissues to cure patients. As of today (Dec 23, 2019), publicly available information (and link to access, among other, to “informed consent in the presence of witnesses” form, may be found, in English, at http://www.idisba.es/en/Support-Services/Scientific-Technical-Platforms/Biobank)

 

- the authors use MT-antibodies, distributed by ABCAM. To my knowledge, it has been notoriously difficult to generate valid MT antibodies. If ABCAM should have been successful, the authors have to validate these antibodies by, for example, showing immunohistochemical validation by comparison of eyes of wildtype and MT-knockout mice.

Answer. Despite attempting it, we have not been able to obtain samples from MT1 or MT2 KO animals. On the other hand, we do not intend to have a colony of those animals. Furthermore, our Institution has implemented a moratory and no more mice colonies can be installed; there are quite a huge number of transgenic animals in the facilities located in the Faculties of the  Univ of Barcelona. We did, however, check specificity of antibodies (ref no. indicated in the revised version) by using untransfected and transfected cells. The results are presented and shown in Annex as figure A3. The specificity results fit with the technical information given by Abcam, the provider of the antibodies here used.

- it remains unclear, why the functional characterization of heteroceptors was moved into Appendix figures, as this is one major conclusion of importance of this study. Notably, these Appendix figures were not accessible to the reviewer.

Answer. We appreciate the comment but we perhaps did not understand the meaning of Appendix. Appendix figures were in the main body, right before the reference list. In addition, other journals it is recommend to show controls in “supplementary material”. In summary, if we are instructed by the Editorial Office we may change locations, e.g. move figs into main text. Also I assure the reviewer that the Figs in Appendix are present in the main document. For sure these figures must be available to reviewers (and if the paper is accepted, to all readers).

 

MINOR COMMENTS

- page 2, line 88: these are not citations that link to the original cloning of the mammalian MTs.

Answer. Thanks for noticing. We have added info on cloning thus referencing Reppert et al., 1994 and 1995

- page 10, line 412: the authors should give a reference for the characterization of IIK7, which is here claimed to be a selective MT2 agonist.

Answer. We have taken this suggestion into account in the revised version of the paper. The ad hoc ref is included (Sugden D, Yeh LK, Teh MT. Design of subtype selective melatonin receptor agonists and antagonists. Reprod Nutr Dev. 1999 May-Jun;39(3):335-44).

- page 12, line 478: the authors should please explain, what is meant with ‘bi-directional cross-antagonism’.

Answer. We have taken this suggestion into account in the revised version of the paper

- page 14, line 535: it is a vast over interpretation, to attribute the pineal gland to be ‘the most important gland in the mammalian body’. This has to be majorly weakened, regarding the adrenal, the pituitary the liver the gut as also important endocrine glands.

Answer. As suggested we have eliminated the indicated words.

Round 2

Reviewer 1 Report

The article is improved.

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