Quantitative Proteomics Reveal Region-Specific Alterations in Neuroserpin-Deficient Mouse Brain and Retina: Insights into Serpini1 Function

Round 1

Reviewer 1 Report

The author has conducted a thorough and comprehensive study on the neuroserpin network within the nervous system, employing a knockout mouse model and tandem mass tag (TMT) analysis. The results were meticulously presented and elucidated, providing a clear understanding of the findings. Additionally, the author delved into a comprehensive discussion regarding the limitations and potential issues associated with the study.

For instance, the author addressed limitations in experimental techniques such as TMT and bottom-up proteomics. Furthermore, the discrepancy in the quantification of Tau protein in the cerebellum (as illustrated in Fig. 6d) between mass spectrometry (MS) data and western blotting (WB) was carefully discussed. Although the author suggested that this discrepancy may stem from various technical factors, it is noteworthy that the consistency between MS and WB results for the other three proteins (Fig. 6a, 6b, 6c) raises questions about the validity of attributing the discrepancy solely to technical factors.

Nevertheless, the manuscript is exceptionally well-written, presenting completed results and engaging in comprehensive and insightful discussions.

Here are two typos need to be correct:

Line 278, the annotation for e) was written by f).

Table 1, Dawn Reg proteins for Retina, is it “Hp” or “HIp”? Feel like there is extra marker on the table. 

Author Response

Thank you for reviewing the manuscript and providing your valuable feedback. We have corrected the typo and removed the mouse cursor mark (the correct gene symbol is Hp) in the final submission.

Author Response File: Author Response.pdf

Reviewer 2 Report

In this article, the authors investigated changes in the proteome of retina and brain tissues in Serpini1 knockout mice using quantitative mass spectrometry-based proteomics with TMT labelling revealing dysregulation in various biological pathways (visual perception, apoptosis, glutamate receptor activation, and supramolecular fibre organization) across different brain regions.

Overall, the article is well-written, with a clear abstract and an introduction providing comprehensive background information on the topic. However, the results are challenging to follow and interpret. Figures often do not align with the text, and legends and labels are too small, making it difficult for readers to distinguish between different samples.

In details:

- Figure 2a is challenging to read and interpret, with unclear correlation between the numbers in the text and those in the figure.

- Figure 2b suffers from poor colour choice and resolution, hindering understanding.

- Figure 2c is unclear, with a tiny legend and unreadable gene references.

- Figure 3 is difficult to read, reporting different numbers from the text, the font size is too small, and reliance on captions to associate the graph with the relevant tissue.

- Figure 4 lacks tissue references, making interpretation impossible without consulting the caption.

- Figure 5 is illegible due to low quality, with unreadable lines and confusing legends (they have opposite orientation, size and style).

- In Figure 6, why does the β-actin of KO mice appear to migrate slower than that of WT mice?

In general, legends for all figures do not sufficiently explain the content.

My suggestion is to review the figures aiming to make them more immediate, precise (clearly reporting the numbers mentioned in the text), and easier to read (with a clear reference above each graph, for example).

Typos:

- Line 242: "retina" is repeated twice.

- Line 263: Space missing before "697."

- Line 289: "up- and down-regulated"

- Line 308: "Increasing" is repeated twice in the same sentence.

- Line 310-311: The phrase "Interestingly, many pathways that showed region-wise dysregulation did appear in STRING. Pr-Pr interaction functional enrichment analysis (Fig. 5)" is unclear.

- Line 344-345: The statement "Complement and coagulation cascade pathway included two members of Serpin family A (Serpina1a and Serpina1e) with a decrease of 0.79 and 0.55-fold, respectively (Fig. 4b)" is unclear as the values of these two proteins are not reported in Fig 4b.

References are used appropriately, and the publication guidelines are respected.

Author Response

Comments and Suggestions for Authors

In this article, the authors investigated changes in the proteome of retina and brain tissues in Serpini1 knockout mice using quantitative mass spectrometry-based proteomics with TMT labelling revealing dysregulation in various biological pathways (visual perception, apoptosis, glutamate receptor activation, and supramolecular fibre organization) across different brain regions.

Overall, the article is well-written, with a clear abstract and an introduction providing comprehensive background information on the topic. However, the results are challenging to follow and interpret. Figures often do not align with the text, and legends and labels are too small, making it difficult for readers to distinguish between different samples.

We sincerely appreciate your review of the manuscript. We've implemented your suggestions and amended all figures to enhance clarity.

In details:

- Figure 2a is challenging to read and interpret, with unclear correlation between the numbers in the text and those in the figure.

While Upset plots generally be a little confusing to interpret, they offer a deeper insight into the relationships between multiple datasets compared to Venn diagrams. This would be particularly valuable for experts interested in proteomic signatures across the analyzed tissues. To enhance clarity, we've labeled the total identified proteins for each condition onto the leftmost color bars in Figure 2a. Additionally, key numbers and labels have been enlarged for better readability.

- Figure 2b suffers from poor colour choice and resolution, hindering understanding.

To improve clarity, we have revised the resolution and color keys. For a better comparison of tissues and samples 2D PCA plots in supplementary files are recommended.

- Figure 2c is unclear, with a tiny legend and unreadable gene references.

Fig 2c has been replaced with an SVG version, allowing for greater zoom-in. Legends and layout have also been modified.

- Figure 3 is difficult to read, reporting different numbers from the text, the font size is too small, and reliance on captions to associate the graph with the relevant tissue.

Fonts were increased in size except redundant text on top of each graph. Note that DEPs are categorized as down- and up-regulated proteins in this fig, with their totals aligning with the main text. Tissue labels have been placed directly on top of each graph to minimize the need for captions.

- Figure 4 lacks tissue references, making interpretation impossible without consulting the caption.

The same labelling was done for Fig 4 with other modifications.

- Figure 5 is illegible due to low quality, with unreadable lines and confusing legends (they have opposite orientation, size and style).

Fig 5 was replaced with a vector-based file format. More information was added to the caption to make it easier for the reader to follow. While this figure may still appear dense, it shows the effects of neuroserpin across all regions within a single visual.

- In Figure 6, why does the β-actin of KO mice appear to migrate slower than that of WT mice?

In general, legends for all figures do not sufficiently explain the content.

That was an intriguing issue however as you can the reverse trend is observed in Tau Western blots! In other western blots (data not provided) we observed a random pattern, likely caused by expired gels. This issue was resolved after using fresh gels.

My suggestion is to review the figures aiming to make them more immediate, precise (clearly reporting the numbers mentioned in the text), and easier to read (with a clear reference above each graph, for example).

Typos:

- Line 242: "retina" is repeated twice.

- Line 263: Space missing before "697."

- Line 289: "up- and down-regulated"

- Line 308: "Increasing" is repeated twice in the same sentence.

- Line 310-311: The phrase "Interestingly, many pathways that showed region-wise dysregulation did appear in STRING. Pr-Pr interaction functional enrichment analysis (Fig. 5)" is unclear.

The sentence and typos were improved.

- Line 344-345: The statement "Complement and coagulation cascade pathway included two members of Serpin family A (Serpina1a and Serpina1e) with a decrease of 0.79 and 0.55-fold, respectively (Fig. 4b)" is unclear as the values of these two proteins are not reported in Fig 4b.

This sentence just provides additional context for fig 4b as it is impossible to include all the protein symbols in this type of enrichment analysis.

References are used appropriately, and the publication guidelines are respected.

Reviewer 3 Report

The article deals with a relevant topic, is well-written, and adequately supports the conclusions.

I consider its publication pertinent with minor changes.

Consider the following bibliography, which could enrich the background:

Gelderblom et al. Deficiency in serine protease inhibitor neuroserpin exacerbates ischemic brain injury by increased postischemic inflammation. PLoS One. 2013 May 3;8(5):e63118. doi: 10.1371/journal.pone.0063118.

Results:

It would be worth describing Figures 1 and 5 in more detail. In Figure 5, it is not easy to appreciate the details and does not seem to contribute much to the study.

Author Response

We highly appreciate the time you spent on this manuscript and your valuable comments. The reference was added to the bibliography. More information for figs 1 and 5 was provided.

Author Response File: Author Response.pdf