Activation of Extracellular Signal-Regulated Kinase 2 and cAMP Response Element-Binding Protein in Cultured Neurons by the Macrocyclic Ellagitannin Oenothein B
Round 1
Reviewer 1 Report
The work presented by the authors is not suitable for the publication.
The study is poor designed and not well conceived. Authors stated in the introduction the non-availability of the Oenothein B in the central nervous system because it may be unable to penetrate the BBB, and only the metabolites ellagic acid and urolithins function in the brain. Thus, based on these premises, why authors chose to explore the activity of Oenothein B? It is conceptually wrong. Data presented have not a rationale. Authors should investigate the activity of the active metabolites.
The investigation of the MAPKs signaling pathway needs the use of inhibitors or antagonist of this pathway as control to confirm the involvement of that pathway.
As a first study screening, the authors should have used an immortalized cell line before using primary cultures involving animal sacrifice.
Author Response
Answer to Reviewer 1
According to the kind suggestion of reviewer 1, we collected our manuscript as follows.
1) About “why authors chose to explore the activity of Oenothein B?”:
As pointed out by reviewer, we have to explain why we added oenothein B, which may be unable to penetrate into the brain (lines 48-51 of original manuscript), to the medium of cultured neurons (lines 70-72 of original manuscript).
In the modified manuscript, we explained this contradiction in Introduction (lines 56-64) while referring References 17 and 18. Correspondingly, we changed the sentences of Abstract (lines 15-17 and lines 20-21).
2) About “… needs the use of inhibitors or antagonist of this pathway as control to confirm the involvement of that pathway.
As pointed out by reviewer, it should be important to investigate whether the inhibitors (such as U0126) inhibit the effect of oenothein B. As we have not yet studied about this problem, we avoided the statement as “activation of the ERK/CREB signaling pathway”. We changed this warding to “activation of ERK and CREB” (line 2 of Title; line 19 of Abstract; line 63 of Introduction; line 131 of Results; line 218 of Discussion). We also added our suggestion in Discussion (lines 252-256).
3) About “the authors should have used an immortalized cell line before using primary cultures involving animal sacrifice.”:
As pointed out by reviewer, we had to sacrifice a few pregnant rats in this study. When we made an experimental plan for this study, we selected the primary culture of neurons instead of established neuronal cell lines, such as Neuro2a or PC12, because we wanted to deal with our previous in vivo study (Plants (2021) 10, 1030) using mouse brain hippocampus. As ERK1/2 and CREB are widely distributed in the brain, we used rat cortical cultures in this study instead of hippocampus. The cortical cultures are consisted of a variety of cell types, but there remain a large fraction of neurons having classical pyramidal neuron morphology. The cortical tissues have the merit that a much greater amount of tissue is available for culture, which is considered to be important for biochemical assays or immunoblot analysis.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments to the authors:
The manuscript Ref. neurosci-1759446, entitled «Activation of ERK/CREB Signaling Pathway in Cultured Neurons by the Macrocyclic Ellagitannin Oenothein B» is a well conducted Communication in a short presentation of brief and preliminary results. The authors have made a well guided approximation of their results.
As a short manuscript it complies with the journal criteria, although the authors should try to include this type of data in their research articles, as all may be complementary with no need of a separate publication.
Author Response
Answer to Reviewer 2
I’m grateful for your response. According to the suggestion of Reviewer 1 and Reviewer 3, we collected our manuscript, and highlighted the parts in yellow.
Author Response File: Author Response.pdf
Reviewer 3 Report
The results presented by the authors completely repeat the results obtained earlier. In my opinion, they do not contribute anything new to the understanding of the action of a substance, since they duplicate the data of in vivo experiments. The authors should more clearly describe the novelty and significance of their work.
Moreover, the quality of some immunoblots is very low and there is no load control.
Author Response
Answer to Reviewer 3
According to the kind suggestion of reviewer 3, we collected our manuscript as follows.
1) About “The authors should more clearly describe the novelty and significance of their work.”:
As pointed out by reviewer, we should clarify the novelty and significance of our work. We thus modified the sentences in Abstract (lines 15-17 and lines 20-21), Introduction (lines 56-64) and Discussion (lines 219-225; lines 236-242) and highlighted these parts in yellow.
2) About “the quality of some immunoblots is very low.”:
As pointed out by reviewer, the quality of immunoblots (perhaps especially Figure 3) is far from adequate. The pERK2/ERK2 ratios and pCREB/CREB ratios are given as means±SEM (n=4 gels), and often does not match the representative photographs of Western blot. I would appreciate if you could understand this situation.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
To speculate the possibility that oenothein B reach the brain passing the BBB, which are the oenothein B levels in the plasma? Authors should at least demonstrate that. This also happens in brain injury conditions as the authors hypothesize. However, this molecule is metabolized before reaching the plasma.
Authors state "A large hydrophilic oenothein B is unable to reach the brain, suggesting that any metabolite(s) of oenothein B might function in the brain", thus authors should investigate before the molecule metabolism after oral administration, then the metabolites should be analyzed, their plasma levels measured and their effect on the tissues investigated.
Alternatively take a measurement of oenotehine B in the brain tissue e.g. by HPLC-MS to see if it is really there.
I wonder on which biological and pharmacological bases the author's previous in vivo work (10.3390/plants10051030) is also based. There is a conceptual mistake!
Author Response
According to the kind suggestion of reviewer 1, we collected our manuscript as follows.
# About the conceptual mistake of our study:
We sincerely apologize that we could not clearly comment the aim of our present study. In the process of modifying the text according to your comments, our explanation is likely to be improving little by little. We are very grateful to you.
As pointed out by reviewer, we have not yet actually confirmed the existence of oenothein B in the brain. We thus mentioned this fact in Line 63 in the revised manuscript. Under such circumstance, we did the present study because we considered that water-soluble oenothein B must directly act on the neurons. In fact, many researchers added oenothein B to the culture medium of various cells in order to investigate their effect(s) and its mechanism(s). We thus added the description in Lines 65-66 and cited two references (Ref19 and Ref 20).
Reviewer 3 Report
The manuscript can be published.
Author Response
I’m grateful for your response.
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
In the manuscript "Activation of the Neuronal Signaling Pathway in the Central Nervous System by Oenothein B, a Bioactive Ellagitannin" Okuyama and colleagues look to extend on their previous work showing that oral administration of the dimeric ellagatanin, oenothein B protected against neuroinflammation by showing the activation of neuronal signalling pathways in the hippocampus of healthy mice fed oenothein B. They attempt to further extend this observation using neuronal cell culture experiments with oenothein B. Collectively, from these experiments they conclude that oenothein B can enhance neuronal signalling pathways and could be neuroprotective.
While the results from the animal experiments, showing that oral administration of oenothein B induces ERK1/2 and CREB signalling pathways in the hippocampus of healthy mice, are interesting the manuscript needs signficant work to improve it to publication standard.
Main comments:
The extreme brevity of both the introduction and discussion means that neither section adequately contextualises the study. Apart from demonstrating that oral administration of oenothein B can alter signalling pathways within the hippocampus of mice in the absence of neuroinflammation it is not clear what gap in current knowledge these experiments are addressing or how the findings from this study increase our knowledge of polyphenols as neuroprotective or anti-inflammatory compounds.
Particularly the disucssion needs signficantly more work to fully explain the significance of the findings from this study within the context of what is currently known about ellagatannins, their metabolites, neuroinflammation and neuroprotection.
It is stated in the introduction that because oenothein B is a large hydrophillic molecule that is unable to cross teh blood brain barrier and ellagitannins are quickly hydrolysed in the small intestine when consumed orally and therefore it is most likely that the neuronal effects are mediated by metabolites of oenothein B rather than the parent compound. It is not clear why oenothein B was used in the in vitro experiments, particularly when there is a plan (mentioned in the dicussion) to identify and test the intestinal metabolites of oenothein B for their ability to modulate ERK1/2 and CREB to demontrate that oenothein B can act as a pro drug.
The assumption that the phenolic hydroxy residue/s on oenothein B are important for ERK1/2 phosphorylation should have been tested and confirmed in cell culture. Alternatively, an in vitro digestion of the parent moecule could have been used in the cell culture to show that oenothein B still retains the ability to phosphorylate ERK1/2 once metabolised.
It would also have been informative for the authors to show that activation of ERK1/2 and CREB within the brain led to measurable change in memory formation through employing some kind of spatial learning test or through measuring changes in long term potentiation within the hippocampus. These experiemtns would strengthen the claim that oenothein B and/or it's metabolites have an impact on neuronal signalling pathways and neuronal function that are physioloically relevant.
Cureently this manuscript has an interesting observation but lacks the additional experiments and scientific context to really explain the importance or relevance of the observation. It perhaps would be better to wait until the proposed additional experiments invesitgating oenothein B metabolites for their ability to cross teh blood brain barrier and activate signalling pathways within the hippocampus are conducted to present a more comprehensive and impactful manuscript.
Minor comments:
The representative western blot images, particularly for p-CREB in Fig 3 and 5 are poor quality and do not appear to clearly show the differences between the different treatments reported in the graphs, better images should be selected.
The representative western blots for both p-ERK-1 and p-ERK-2 and total ERK-1 and ERK-2 are presented (figure 2 and 4) but only the graphs of the ratio between p-ERK-2 and total ERK-2 are presented and discussed. Particularly fig 4B where it appears that p-ERK-1 peaks at 10 mins before declining, unlike p-ERK-2 which is still elevated at 30 mins post oenothein B treatment, it would interesting to examine these findings and contextualise them either in the context of neuronal signalling or immune modulation.
Reviewer 2 Report
This is a scientifically solid work. The manuscript is well written and the conclusions are supported by the data. This manuscript merits publication.
Reviewer 3 Report
The aim of this small observational study was to investigate effects of oenothein B on the activation of ERK1/2 and CREB pathways in vitro and in vivo.
Here are some suggestions for the improvement, at least in vitro:
- The authors did not demonstrate that CREB activation is driven by ERK-signalling. They are suggested to use UO126 or some other suitable inhibitor of ERK signalling and to monitor effects on CREB phosphorylation to investigate if CREB activation is mediated by ERK.
- It would be interesting to monitor activation of other signalling pathways related to CREB activation: PI3K/Akt/GSK-3β, PKA, CaMKII,….
- Iit would be interesting to investigate effects on ERK/CREB signalling in oxidative stress-conditions.
- On Page 5, line 176 the authors wrote: ...... suggesting that it takes more than 3 days to exert its effects in the brain; i.e., for the intestinal hydrolysisof oenothein B, the translocation of its metabolite(s) across physiological barriers, such as the gastroenteric barrier or BBB, and the mediation of signal transduction in the brain...... in order to explain why effect of oenothien B on the pERK/ERK ratio was not observed on day 3.
As authors observed that oenothein B was capable to exert protective neuroinflammatory response after 3 days of administration (ref.8), this explanation seems unlikely.
Minor issues
Page 2, line 63 - also known to play an important regulator of the expression of glial cell line-derived neurotrophic factor [18] – changed to ....also known to play an important regulatory role in the expression
Page 5, lines 188 – 192; already explained in the introduction
Page 5, lines 193-197: it is not clear why antioxidative activity was mentioned as OS parameters were not investigated.
