Effects of Stathmin 1 Gene Knockout on Behaviors and Dopaminergic Markers in Mice Exposed to Social Defeat Stress

Round 1

Reviewer 1 Report

At first sight his paper may appear as a rather traditional phenotyping study of a new genetically manipulated model. However, the very accurate choice of behavioral protocol as well as the thoughtful data analyses  render this work and its findings interesting and stimulating beyond the specific goal of the study.

I especially appreciated the use of a social interaction test beside the more standard social avoidance test. 

For this reason I would have been a little more speculative in the discussion pointing to the general implication of the relationship (or lack of it) between the indications derived by the different tests.

As an example: is it possible that reduced exploration of the novel object by  STMN1 KO mice is due to novelty avoidance  dependent on high anxiety? 

Moreover, what are the implications of the observed lack of relationship between reduced social exploration expressed by STMN1 KO mice in the standard test and in the social interaction test ?

Anyhow, I leave to the authors the choice to accept this suggestion or leave the discussion as it stands

Author Response

Comment. We thank the editor and reviewers for the constructive criticism. The revised manuscript has more focused in method and conclusion. We also present the consider and findings in a clearer manner according to the suggestions of the reviewers. In particular:

Review 1: Comments and Suggestions for Authors

At first sight his paper may appear as a rather traditional phenotyping study of a new genetically manipulated model. However, the very accurate choice of behavioral protocol as well as the thoughtful data analyses render this work and its findings interesting and stimulating beyond the specific goal of the study. I especially appreciated the use of a social interaction test beside the more standard social avoidance test. For this reason I would have been a little more speculative in the discussion pointing to the general implication of the relationship (or lack of it) between the indications derived by the different tests.

Point 1: As an example: is it possible that reduced exploration of the novel object by STMN1 KO mice is due to novelty avoidance dependent on high anxiety? 

Response) It could be due to high anxiety because STMN1 KO mice spent less time in the central zone in the open field test. Your suggestion is valuable but exploration the relationship between different behavioral tests was not our objectives.

Point 2: Moreover, what are the implications of the observed lack of relationship between reduced social exploration expressed by STMN1 KO mice in the standard test and in the social interaction test? Anyhow, I leave to the authors the choice to accept this suggestion or leave the discussion as it stands.

Response) If you may, we would like to leave the discussion as it stands because relationship between the tests was not our main objectives.

Reviewer 2 Report

In the manuscript entitled “Effects of Stathmin 1 Gene Knockout on Behaviors and Dopaminergic Markers in Mice Exposed to Social Defeat Stress” by Nguyen et al, the authors have shown the relevance of stathmin1 to behavioral parameters in mice exposed to stress associated with social defeat. Social stress has major impact on mental and psychological health especially in today’s scenario; therefore this is an important study. However there are several concerns.

The role of Stathmin 1 in social behavior associated stress is not new. Prior studies have shown that Stathmin is a well-known modulator of psycho social behavior and deficiency of Stathmin has been shown to be associated with deficiency in innate and learned fear, in assessing the stress and threats.

Previous work by the same group has shown the association of brain dopamine receptors especially dopamine D2 receptors and other dopaminergic markers with psycho social behavior and also with social defeat associated stress. In this study, the authors have tried to link Stathmin 1 to dopaminergic alteration associated with social defeat stress. However, the link is not clear. The study is inconclusive and it fails to demonstrate a clear connection between STMN 1 and dopaminergic alteration. Differences are not clearly evident from the images provided, especially the western blots.

For e.g. Figure 5: From WB images provided it looks like that only changes occur in STMN1 phosphorylation in the PFC region and not in any other regions.  However, it is not clear how this Stathmin activity change is associated with dopaminergic alteration and behavioral modulation.

Moreover, why were expressions of total Stathmin 1 and phospho Stathmin 1 given in two separate figures?

Immunohistochemical staining of Stathmin 1 (Figure 5C) is not clear and staining quality is very poor. Slides should preferably be counterstained or histopathological staining of the slides must be provided.

The experiments lack proper controls. What is the status of Stathmin 1 in SDS group compared to unchallenged control group? Is there any alteration associated with Stathmin 1 expression or activity in these SDS mice compared to control mice?

The results were presented in a confusing manner which comparison among different groups difficult. Figure 2 the comparison among groups are not clearly mentioned.

Figure 3 ,graphical representation of effect of social defeat on recognition Index between wild type and knockout mice: how was this calculated? It doesn’t look very different among wild and KO groups, both before and after the stress. However, the authors have labelled this as statistically significant.

References need to be checked and corrected. There are numerous typos in the author’s name section, including their own reference.

Author Response

Response to Reviewer 2 Comments

Comment. We thank the editor and reviewers for the constructive criticism. The revised manuscript has more focused in method and conclusion. We also present the consider and findings in a clearer manner according to the suggestions of the reviewers. In particular:

Review 2: Comments and Suggestions for Authors

In the manuscript entitled “Effects of Stathmin 1 Gene Knockout on Behaviors and Dopaminergic Markers in Mice Exposed to Social Defeat Stress” by Nguyen et al, the authors have shown the relevance of stathmin1 to behavioral parameters in mice exposed to stress associated with social defeat. Social stress has major impact on mental and psychological health especially in today’s scenario; therefore this is an important study. However there are several concerns. The role of Stathmin 1 in social behavior associated stress is not new. Prior studies have shown that Stathmin is a well-known modulator of psycho social behavior and deficiency of Stathmin has been shown to be associated with deficiency in innate and learned fear, in assessing the stress and threats.

Point 1: Previous work by the same group has shown the association of brain dopamine receptors especially dopamine D2 receptors and other dopaminergic markers with psycho social behavior and also with social defeat associated stress. In this study, the authors have tried to link Stathmin 1 to dopaminergic alteration associated with social defeat stress. However, the link is not clear. The study is inconclusive and it fails to demonstrate a clear connection between STMN 1 and dopaminergic alteration. Differences are not clearly evident from the images provided, especially the western blots. For e.g. Figure 5: From WB images provided it looks like that only changes occur in STMN1 phosphorylation in the PFC region and not in any other regions.  However, it is not clear how this Stathmin activity change is associated with dopaminergic alteration and behavioral modulation.

Response) We added more discussion as follows plus new figure 8.

“Possible mechanisms may be speculated as follows (Figure 8). Dopamine (DA) released in response to SDS may bind to D1 receptors and increase pDARPP-32 Thr34 via protein kinase A (PKA) activation. At the same time, DA may bind to D2 receptors and increase pDARPP-32 Thr75 via cyclin-dependent kinase 5 (Cdk 5) activation 5 [1]. This also leads to reduction of pDARPP-32 Thr34 via inhibition of PKA. Given that D1 receptor expression was reduced in the frontal cortex of defeated mice [2], a net effect on pDARPP-32 Thr34 would be zero or inhibition. As reduction of pDARPP-32 Thr34 can lead to increased activation of PP-1 [1,3,4] and stathmin at Ser16 is known to be dephosphorylated by PP-1, PP-2A, and PP-2B [5,6] final effect of SDS would lead to decrease of pSTMN-16 in the PFC of defeated mice.”

Point 2: Moreover, why were expressions of total Stathmin 1 and phospho Stathmin 1 given in two separate figures?

Response) We measured pSTMN1-16 and total STMN on separate gels because we were concerned of getting reliable signal of the total STMN1 after membrane stripping. Because of this limitation, we did not use the ratio (phospho/total). Nevertheless, to ensure reliability, we ran the samples in duplicate. Of note, several recent studies on Darpp32 [7,8] have reported total and phospho in two separate figures.

Point 3: Immunohistochemical staining of Stathmin 1 (Figure 5C) is not clear and staining quality is very poor. Slides should preferably be counterstained or histopathological staining of the slides must be provided.

Response) We replaced the figure with more higher resolution pictures.  

Point 4: The experiments lack proper controls. What is the status of Stathmin 1 in SDS group compared to unchallenged control group? Is there any alteration associated with Stathmin 1 expression or activity in these SDS mice compared to control mice? The results were presented in a confusing manner which comparison among different groups difficult. Figure 2 the comparison among groups are not clearly mentioned.

Response) We clearly described the results of total and phosphorylated STMN1 levels in the Figure 5 (A and B). In the figure 2, we described the results of comparison among groups only when significant interaction was found. Please refer to supplementary table 1 for more detailed analysis. 

Point 5: Figure 3, graphical representation of effect of social defeat on recognition Index between wild type and knockout mice: how was this calculated? It doesn’t look very different among wild and KO groups, both before and after the stress. However, the authors have labelled this as statistically significant.

Response) We only described the results of two way ANOVA in the figures. Therefore, meaning of significance for genotype is that there is difference between the genotypes regardless of phenotypes (unsusceptible or susceptible). For more details, refer to supplementary table 2.

Point 6: References need to be checked and corrected. There are numerous typos in the author’s name section, including their own reference.

Response) We corrected the related parts. Many thanks.

References

Reis, H.J.; Rosa, D.V.; Guimarães, M.M.; Souza, B.R.; Barros, A.G.; Pimenta, F.J.; Souza, R.P.; Torres, K.C.; Romano-Silva, M.A. Is DARPP-32 a potential therapeutic target? Expert Opinion on Therapeutic Targets 2007, 11, 1649-1661. Avgustinovichi, D.; Alekseyenko, O. [3 H] SCH 23390 Binding in Various Brain Regions of C57BL/6J Mice with Repeated Experience of Victory or Social Defeat in Agonistic Interactions. Physiological research 2010, 59. Nishi, A.; Shuto, T.J.E.o.o.t.t. Potential for targeting dopamine/DARPP-32 signaling in neuropsychiatric and neurodegenerative disorders. Expert Opinion on Therapeutic Targets 2017, 21, 259-272. Greengard, P.; Allen, P.B.; Nairn, A.C.J.N. Beyond the dopamine receptor: the DARPP-32/protein phosphatase-1 cascade. Neuron 1999, 23, 435-447. Machado-Neto, J.A.; Saad, S.T.O.; Traina, F.J.B.r. Stathmin 1 in normal and malignant hematopoiesis. BMB Reports 2014, 47, 660. MISTRY, S.J.; Heng-Chun, L.; ATWEH, G.F.J.B.J. Role for protein phosphatases in the cell-cycle-regulated phosphorylation of stathmin. Biochemical Journal 1998, 334, 23-29. . Femenia, T.; Qian, Y.; Arentsen, T.; Forssberg, H.; Heijtz, R.D. Toll-like receptor-4 regulates anxiety-like behavior and DARPP-32 phosphorylation. Brain, behavior, and immunity 2017.

8.        López-Cruz, L.; San Miguel, N.; Carratalá-Ros, C.; Monferrer, L.; Salamone, J.D.; Correa, M.J.N. Dopamine depletion shifts behavior from activity based reinforcers to more sedentary ones and adenosine receptor antagonism reverses that shift: relation to ventral striatum DARPP32 phosphorylation patterns. Neuropharmacology 2018, 138, 349-359.

Round 2

Reviewer 2 Report

The authors have answered all the queries.