Precise Regulation of the Basal PKCγ Activity by DGKγ Is Crucial for Motor Coordination

Round 1

Reviewer 1 Report

In this manuscript, the authors show the function of DGKgamma in cerebellum using Purkinje cell-specific knockout mice. As a mechanism of it, the absence of dissociation of GluR2 and GRIP during chemically induced LTD in those mice was revealed, which led to the impairment of LTD and deficit of motor function. Overall, they are showing an interesting and novel experimental data, however, I have some concerns about the reliability of them.

Major

1. In figure 1, it is difficult to understand how the PCR genotyping works. Please add the primer position to fig 1a.

2. In fig3&4, the authors have done the IP experiment on GRIP and TRPC3. The same experiment using control IgG should be done at least on vehicle groups, because the reliability of precipitated proteins is low without that. Especially, the phosphorylation of TRPC3 detected by anti-PKC substrate antibody is not convincing by only present data.

Minor

1. In Page 2, line 49, the reference [5] should be [15].

2. In Page 3, the legend of Fig2, WT and KO should be replaced to tm1c and tm1d, respectively.

Author Response

Thank you for your constructive comments. According to your suggestions and comments, we revised the manuscript as follows.

To major comments

1. We revised Fig. 1 and added the primer position in it.
2. For Figs. 3 & 4, we performed additional IP experiments using control IgG, and added the data showing the specificity of respective antibody.

To minor comments

1. We revised the reference number.
2. We revised the legend of Fig. 2.

In addtion, "materilas & methods" and "results" were improved. The sentence we changed or added are shown in red.

Reviewer 2 Report

Tsumagari et al presented a detailed study on the regulation of PKC involved in the LTD through the activity of DGK. This study is novel in many ways and will contribute immensely in understanding the molecular basis of  cerebellar long-term depression and will facilitate in the development of new therapeutic strategies regarding the long term depression. The manuscript is acceptable in current form for the publication. 

Author Response

Thank you for your kind comments. According to your and other reviewer's suggestions commetns, we revised the manuscript. The sentences we changed and added are shown in red.

Reviewer 3 Report

The present manuscript extends the results of a previous work by the same group, in which the authors described the role of DGKγ in CNS using DGKγ KO mice. In this paper the authors generated Purkinje cell-specific DGKγ KO mice (tm1d), and observed cerebellar motor dysfunction and impaired LTD similar to that obtained in DGKγ KO model. Moreover, the data in this manuscript demonstrated that basal PKCγ is up-regulated in tm1d mice, and that using PKCγ inhibitor the rescue of LTD occurs. The subject of the manuscript is interesting and the paper is well written and well discussed. However, there are some points that need to be addressed to make this study more sound.

Specific points:

The Electrophysiological properties of Purkinje cells in tm1d mice were not reported, even if in the previous study the authors reported this information for DGKγ KO mice. This would have characterized tmd1 mouse model and should be addded.
Figure 4 is of poor quality: the bands are very faint and barely detectable. A new figure with more convincing images is needed.

Author Response

Thank you for your constructive comments. According to your suggestions and comments, we revised the manuscript as follows.

1. As you pointed out, LTD in Tm1d mouse should be presented. However, we don’t have enough time to do it. We think that LTD in tm1d mouse is impaired based on similar phenotype of the conditional KO mice with the tm1a KO mice.
2. We revised the Fig. 4.

In addition, "materials & methods" and "results" were improved. The sentences we changed or added are shown in red.

Round 2

Reviewer 1 Report

The authors have revised manuscript properly. Now, this is fully acceptable for the journal.

Reviewer 3 Report

Even if, electrophysiological analyses would have added soundness to the results, I consider the quality of the manuscript enough good to deserve to be published on International Journal of Molecular Sciences.