Neuronal Excitation Induces Tau Protein Dephosphorylation via Protein Phosphatase 1 Activation to Promote Its Binding with Stable Microtubules

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Neurolint-3011456. An interesting work regarding physiology and pathology of tau protein. I have questions, suggestions and reservations about the manuscript. In the title of the work should be tau protein not tau. Tau is a letter of the Greek alphabet and it is difficult to study this letter in culture or in the animal brain. The correct term tau protein should be used throughout the manuscript. Testing tau in vitro and in vivo is scientific jargon and an ordinary reader will certainly be surprised when reading the text - how is it possible?! In sentence lines 26-28, should be added changes in tau protein after brain ischemia (Int J Mol Sci. 2024 Mar 7;25(6):3091. doi: 10.3390/ijms25063091.). The methods should be supplemented with the date of consent of the bioethics committee, the species/breed of mice, their number, and age and body weight. For reagents or equipment, please provide the country and city in addition to the company. Figure 1A what units are on the vertical axis? Figure 1 add used test. P. 5, line 171 what it is NE? Figure 2B,C what units are on the vertical axis? Figure 2 add used test. Discussion P.6, line 215 add suggested literature after hypoxia. P.6, line 234 add after conditions citation. P. 7, line 250 complete with the statement "and post-ischemic brain neurodegeneration".

Author Response

Neurolint-3011456. An interesting work regarding physiology and pathology of tau protein. I have questions, suggestions and reservations about the manuscript. In the title of the work should be tau protein not tau.

Tau is a letter of the Greek alphabet and it is difficult to study this letter in culture or in the animal brain. The correct term tau protein should be used throughout the manuscript. Testing tau in vitro and in vivo is scientific jargon and an ordinary reader will certainly be surprised when reading the text - how is it possible?! 

We changed the term “tau” to “tau protein” as suggested.

 

In sentence lines 26-28, should be added changes in tau protein after brain ischemia (Int J Mol Sci. 2024 Mar 7;25(6):3091. doi: 10.3390/ijms25063091.).

While we do appreciate the suggestion, the review article mentioned by the Reviewer 1discusses the role of tau phosphorylation in the context of brain ischemia, and we thought that it may not be appropriate as a refence for neuronal activity-induced phosphorylation. Instead, we cite this article in the Discussion as mentioned below.

 

The methods should be supplemented with the date of consent of the bioethics committee, the species/breed of mice, their number, and age and body weight.

We added the information (the date of consent of the bioethics committee, strain name, number of mice, age, body weight of the mice) in the Methods section.

 

For reagents or equipment, please provide the country and city in addition to the company.

We added the requested information in the corresponding parts of the Methods section.

 

Figure 1A what units are on the vertical axis?

The bar graphs show the expression levels relative to the control, as explained in the legend. We added “Ratio” to the y axis.

 

Figure 1 add used test.

Two-way ANOVA followed by Sidak’s multiple comparison test was used for statistical analysis. We added this information to the Methods section.

 

1. 5, line 171 what it is NE?

We replaced NE with “novel environment”.

 

 Figure 2B,C what units are on the vertical axis?

The graphs show the quantified levels of AT8 immunoreactive signal relative to the time-zero. We added “Ratio” to the y axis of the graphs.

 

Figure 2 add used test.

The following tests were used for statistical analysis: one-way ANOVA followed by Dunnett’s multiple comparison test (2A)，two-way ANOVA followed by Dunnett’s multiple comparison test(2B, C)，two-way ANOVA followed by Tukey’s multiple comparison test(2D). These explanations are now added to the legend of the figure.

 

Discussion P.6, line 215 add suggested literature after hypoxia. P.6, line 234 add after conditions citation. P. 7, line 250 complete with the statement "and post-ischemic brain neurodegeneration".

We added the suggested reference to the corresponding parts of the Discussion section.

Reviewer 2 Report

Comments and Suggestions for Authors

 

I have read with interest the manuscript titled “Neuronal excitation induces tau dephosphorylation via protein phosphatase 1 activation to promote its binding with stable microtubules” by Yagishita et al. The authors employ mouse primary cortical neuronal cultures and in vivo models to associate the membrane depolarisation with the dephosphorylation of tau and therefore the tighter binding on the microtubules. In the primary cultures they induce this by KCl treatment, a well-established method in the literature, while for the in vivo systems this is achieved by exposure to a new environment.

 

Overall, this is an interesting study with nicely presented results. My main concern is that perhaps the statements are slightly too bold to be supported by the results, especially the ones about the PP1 and its claimed specificity or induction of activity. These claims are essentially supported only by an in vitro phosphatase assay, which is not very well explained in the text. Additionally, in the discussion the authors mention that PP1 activity is induced by KCl-mediated depolarisation in the primary cultures, an experiment that is not presented in the current form of the manuscript. The authors need to revise the manuscript before proceeding with publication.

 

Please see bellow my detailed comments:

 

Line 10: missing “the”

 

The abstract is very badly written with several grammar and syntax mistakes. The authors should address this.

 

A very important question that should be addressed is about the experimental in vivo system: are these transgenic mouse models expressing a human tau isoform or just wild type mice? If yes, which model is this (it needs to be properly cited), which human tau isoform do they express and are there any mutations? Additionally, which strain of mice did the authors use? It is not very nice that they have a method section about animals without mentioning these pieces of information.

 

Line 135: The authors mention that the AT8 antibody is reactive against the following epitopes: S199, S202, and T205. Even though there are reports about immunoreactivity against the S199, in the initial characterisation paper (PMID: 7624036) it is demonstrated that S199 is not within the phosphoepitopes that are recognised by the AT8 antibody. This should be corrected in the manuscript.

 

I think that my main concern is around the phosphatase assay. If I understood correctly from the methods description, the lysates are incubated at 37oC with or without the mentioned inhibitors. This means that the lysates are getting dephosphorylated by the endogenous phosphatases that are included in the mixture, unless they are blocked from the corresponding inhibitor.

      I.         Why the exposure to a new environment should be activating specifically the PP1 and not just simply reducing the activity of the other phosphatases?

     II.         Have the authors checked whether the levels of PP1 are upregulated under these experimental conditions?

   III.         How do the authors claim that PP1 specifically dephosphorylates the AT8 epitopes, without testing other phospho-tau antibodies such as the ones used in Figure 1?

   IV.         Additionally, since the authors are connecting the exposure of the new environment with the KCl-dependent depolarisation in the culture experiments, they should perform the phosphatase assay in the KCl-depolarised neurons and compare the findings with the in vivo data.

    V.         Tautomycetin also demonstrates an inhibitory activity against PP2A, but with lower affinity (PMID: 11554729), which should be mentioned in the manuscript

 

Line 219: the authors claim that KCl stimulation induces the activity of PP1. This claim is false or at least this is not shown in any of the presented data and therefore the authors should either provide the results to support this claim or revise the sentence.

 

Line 241-242: I am not a supporter of the phrase “data not shown”. Nowadays publishing groups allow great flexibility to include data and I encourage the authors to include these results in this manuscript, even if they are negative data.

Comments on the Quality of English Language

The authors should improve the quality of the English language in the abstract.

Author Response

I have read with interest the manuscript titled “Neuronal excitation induces tau dephosphorylation via protein phosphatase 1 activation to promote its binding with stable microtubules” by Yagishita et al. The authors employ mouse primary cortical neuronal cultures and in vivo models to associate the membrane depolarisation with the dephosphorylation of tau and therefore the tighter binding on the microtubules. In the primary cultures they induce this by KCl treatment, a well-established method in the literature, while for the in vivo systems this is achieved by exposure to a new environment.

 

Overall, this is an interesting study with nicely presented results. My main concern is that perhaps the statements are slightly too bold to be supported by the results, especially the ones about the PP1 and its claimed specificity or induction of activity. These claims are essentially supported only by an in vitro phosphatase assay, which is not very well explained in the text. Additionally, in the discussion the authors mention that PP1 activity is induced by KCl-mediated depolarisation in the primary cultures, an experiment that is not presented in the current form of the manuscript. The authors need to revise the manuscript before proceeding with publication.

 

Please see bellow my detailed comments:

 

Line 10: missing “the”

 "The" is added to the line 10.

The abstract is very badly written with several grammar and syntax mistakes. The authors should address this.

 The abstract text has been modified.

A very important question that should be addressed is about the experimental in vivo system: are these transgenic mouse models expressing a human tau isoform or just wild type mice? If yes, which model is this (it needs to be properly cited), which human tau isoform do they express and are there any mutations? Additionally, which strain of mice did the authors use? It is not very nice that they have a method section about animals without mentioning these pieces of information.

 C57BL/6J wild type mice were used for in vivo experiments in the described work. This information is now added to the Methods section.

Line 135: The authors mention that the AT8 antibody is reactive against the following epitopes: S199, S202, and T205. Even though there are reports about immunoreactivity against the S199, in the initial characterisation paper (PMID: 7624036) it is demonstrated that S199 is not within the phosphoepitopes that are recognised by the AT8 antibody. This should be corrected in the manuscript.

 In response to the comment, we deleted the description on S199.

I think that my main concern is around the phosphatase assay. If I understood correctly from the methods description, the lysates are incubated at 37oC with or without the mentioned inhibitors. This means that the lysates are getting dephosphorylated by the endogenous phosphatases that are included in the mixture, unless they are blocked from the corresponding inhibitor.

1. Why the exposure to a new environment should be activating specifically the PP1 and not just simply reducing the activity of the other phosphatases?

We found that the reduction of AT8 site phosphorylation is likely mediated by PP1 activity, because the PP1 inhibitor prevented the reduction of AT8 site phosphorylation.  We would like to point out that involvement of phosphatases other than PP1 cannot be fully ruled out by our experiments.  Our data only show that the inhibitors for PP2A or PP2C do not prevent the reduction of AT8 site phosphorylation in the experimental condition that we used.

1. Have the authors checked whether the levels of PP1 are upregulated under these experimental conditions?

We quantified the PP1 protein expression levels in the mouse brain 5min, 30 min, 1h, 24hr after initiation of novel environment exposure to find that PP1 protein expression levels are comparable in all the examined time points.

   III.         How do the authors claim that PP1 specifically dephosphorylates the AT8 epitopes, without testing other phospho-tau antibodies such as the ones used in Figure 1?

As described in the Results section, we chose to examine AT8 sites as a representative phosphorylation sites of tau protein, and our observation is that the PP1 inhibitor prevented the reduction of AT8 phosphorylation. Here we do not try to insist that PP1 specifically dephosphorylates the AT8 epitopes, and our results are not affected by the changes in phosphorylation status of other phosphorylation sites.

IV. Additionally, since the authors are connecting the exposure of the new environment with the KCl-dependent depolarisation in the culture experiments, they should perform the phosphatase assay in the KCl-depolarised neurons and compare the findings with the in vivo data.

We did not perform the suggested experiment. Previous work by Gómez-Ocádiz et al., 2022, which we cited in our manuscript, showed that novel environment exposure induces neuronal depolarization in the mouse brain. We performed the KCl-induced neuronal depolarization experiment based on this report.

As we describe as an answer to the Reviewer 3, we performed the experiment in which we added tautomycetin and/or okadaic acid (inhibitors to PP1 and PP2A) to the neuronal culture and performed KCl stimulation. We found that the addition of the inhibitor(s) certainly affects the AT8 site phosphorylation, but we do not think we can conclude anything out of this experiment, because the phosphatase inhibitors could affect any step between KCl-induced neuronal depolarization and phosphorylation of tau protein, (for instance, the inhibitors could modify sensitivity to KCl). Therefore, we did not include the data in the manuscript.

V. Tautomycetin also demonstrates an inhibitory activity against PP2A, but with lower affinity (PMID: 11554729), which should be mentioned in the manuscript

 We added the explanation about the affinity of tautomycetin to PP2A in the corresponding part of the Methods section, as suggested.

Line 219: the authors claim that KCl stimulation induces the activity of PP1. This claim is false or at least this is not shown in any of the presented data and therefore the authors should either provide the results to support this claim or revise the sentence.

 We deleted the description that PP1 activity is induced by KCl stimulation in culture.

Line 241-242: I am not a supporter of the phrase “data not shown”. Nowadays publishing groups allow great flexibility to include data and I encourage the authors to include these results in this manuscript, even if they are negative data.

Since this description is in Discussion section, and we do not think that the negative data add much to the manuscript, we deleted this paragraph from the Discussion.

Reviewer 3 Report

Comments and Suggestions for Authors

The authors study the neuronal excitation induces tau dephosphorylation in cultured neurons and mice models. They identify that protein phosphatase 1 activation account for tau dephosphorylation, which promote tau protein binding with stable microtubule.  The tau protein aggregation and formation of neuronal tangle is the pathological features of AD. The current study provide new information which can lead to new intervention strategy against neurodegeneration in AD.

However, there are some concerns:

1, In figure brain lysates are used to check the PP1 inhibitor effects on Tau phosphorylation. In Figure 1A, the authors should also use PP inhibitors to cultured neurons and check the phosphorylation status of tau protein in cultured neurons. 

2, in Figure 2B and 2C, no illustration of Y-axis is shown. The authors should add the details for Y-axis

3, Hyperphosphorylation of tau protein can lead to tau protein aggregation. Although the author shows dephosphorylation of Tau. However, the authors should check the culture neurons and mice models whether tau protein aggregation has been affected by KCl-induced neuronal depolarization and Exposure to novel environment in neuron and mice models.  

Author Response

The authors study the neuronal excitation induces tau dephosphorylation in cultured neurons and mice models. They identify that protein phosphatase 1 activation account for tau dephosphorylation, which promote tau protein binding with stable microtubule.  The tau protein aggregation and formation of neuronal tangle is the pathological features of AD. The current study provide new information which can lead to new intervention strategy against neurodegeneration in AD.

However, there are some concerns:

1, In figure brain lysates are used to check the PP1 inhibitor effects on Tau phosphorylation. In Figure 1A, the authors should also use PP inhibitors to cultured neurons and check the phosphorylation status of tau protein in cultured neurons. 

In fact, we performed the suggested experiment; we added tautomycetin and/or okadaic acid to the neuronal culture and performed KCl stimulation. We found that the addition of the inhibitor(s) certainly affects the AT8 site phosphorylation, but we do not think we can conclude anything out of this experiment, because it is possible that the phosphatase inhibitors affect any step between KCl-induced depolarization and phosphorylation of tau protein, (for instance, the inhibitors could modify sensitivity to KCl). Therefore, we did not include the data in the manuscript.

2, in Figure 2B and 2C, no illustration of Y-axis is shown. The authors should add the details for Y-axis

The graphs show the quantified levels of AT8 immunoreactive signal relative to the time-zero. We added “Ratio” to the y axis of the graphs.

3, Hyperphosphorylation of tau protein can lead to tau protein aggregation. Although the author shows dephosphorylation of Tau. However, the authors should check the culture neurons and mice models whether tau protein aggregation has been affected by KCl-induced neuronal depolarization and Exposure to novel environment in neuron and mice models.  

We used wild-type C57BL/6J mice for the described animal experiments. It has been known that mouse tau protein does not form aggregates. Therefore, the possibility that neuronal depolarization may affect tau protein aggregation is beyond the scope of the current work.

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Clarifications related to Figures 1 and 2 regarding the statistical tests used are necessary. The authors write that Figure 1 uses the Sidak's test and Figure 2 uses Dunnett's test. However, the methods also include the Tukey's and Student's test, which is true and what test was actually used. Moreover, the word tau is used in the figures 1 and 2. To solve this problem, in the figure caption you should explain that tau it is tau protein.

Author Response

Clarifications related to Figures 1 and 2 regarding the statistical tests used are necessary. The authors write that Figure 1 uses the Sidak's test and Figure 2 uses Dunnett's test. However, the methods also include the Tukey's and Student's test, which is true and what test was actually used. Moreover, the word tau is used in the figures 1 and 2. To solve this problem, in the figure caption you should explain that tau it is tau protein.

 

Student’s t-test was used for the analysis shown in Figure 1A, and Tukey’s test was used for the analysis shown in Figure 2. We added the description on statistical analysis to the legends of the corresponding figures.

We added the description in the legends of the figures that “Tau” stands for “tau protein”.

 

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have revised their study with significant improvements. However, some concerns still have not been addressed properly. 

1,  although PP1 inhibitors may affect cells response sensitivity to KCl, usage of multiple PP1 inhibitors can provide strong supporting data. So the authors are requested to provide the data of PP1 inhibitors. The PP1 inhibitors data is important and inevitable for their findings.

2, Although mice Tau can not form aggregation, cultured human neuronal model can be applied. The Tau hyperphosphorylation can lead to Tau protein aggregation, which is vital to neurodegeneration in AD. If hyperphosphorylation has no influence on Tau protein aggregation and neurodegeneration, then the significance of findings of the current study will drop dramatically. 

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

The revised version of the manuscript has been improved significantly. All major concerns have been addressed properly. So it can be accepted for publication now.