Cellular Organelle-Related Transcriptomic Profile Abnormalities in Neuronopathic Types of Mucopolysaccharidosis: A Comparison with Other Neurodegenerative Diseases

Round 1

Reviewer 1 Report

This is an excellent paper thoroughly describing different methods of biomarker discovery in MPS disorders. While each form of MPS is rare, taken together they are not that rare and the knowledge of biomarkers is clearly needed to diagnose and assess treatment success. I only found a few issues.

Line 82: diagnosis is not necessarily difficult for all of them. 

Line 118: wouldn’t you worry that the fetal bovine serum includes the missing enzymes partially corrects the cells?

There are a few sentences that need to be reworded for clarity but I will  leave that to the editor, whose English is probably better than mine.

Author Response

This is an excellent paper thoroughly describing different methods of biomarker discovery in MPS disorders. While each form of MPS is rare, taken together they are not that rare and the knowledge of biomarkers is clearly needed to diagnose and assess treatment success. I only found a few issues.

Line 82: diagnosis is not necessarily difficult for all of them. 

RESPONSE:

Indeed, this sentence was not precise. Our intention was to indicate that both the treatment and diagnosis of neurological symptoms in patients with MPS are difficult. This sentence has been corrected and now looks like this (lines 87-89 in the revised manuscript):

“Both treatment and diagnosis of neurological symptoms in MPS patients are difficult due to the lack of reaching the central nervous system (CNS) with current therapies and the lack of markers of neurodegeneration.”

Line 118: wouldn’t you worry that the fetal bovine serum includes the missing enzymes partially corrects the cells?

RESPONSE:

We are aware on this problem. However, when MPS cells were cultured in the medium containing FBS, they were still characterized by reduced activity of selected lysosomal enzymes and increased levels of GAGs. We confirmed this in our previously works (performed with exactly the same MPS cell lines), for example: Wiśniewska et al. (2022) Gene 812:146090; Gaffke et al. (2021) Int J Mol Sci. 22(5):2766.

Reviewer 2 Report

The article shows relationship between transcriptome (but also inferred proteins and other macromolecules) and specific type of MPS. The introduction and methods-approaches are clearly presented.  I suggest improvement in discussion and conclusions. 

a) Discussion should be enriched to be clear in the relationship of the genes-transcripts-proteins for each MPS.

b) The details from microscopy should be clearly linked to protein and mucopolisacharides.

c) Also, additional information should be carefully revised and discussed about other neurological diseases and manifestations to avoid speculative sentences.

d) Scope and limitations of the study should be clearly sentenced. As authors declared just prospective ideas regarding extension of application in other cells and patients.  

e) Conclusions should be specific about the genes-proteins key in each MPS (some data are in the abstract, but no in the conclusions section). Some other -complementary- sentences should be at least in other paragraph. 

English is fine. Minor issues in grammar. 

Author Response

The article shows relationship between transcriptome (but also inferred proteins and other macromolecules) and specific type of MPS. The introduction and methods-approaches are clearly presented.  I suggest improvement in discussion and conclusions. 

a) Discussion should be enriched to be clear in the relationship of the genes-transcripts-proteins for each MPS.

RESPONSE:

We thank the reviewer for this comment. The discussion regarding individual genes and the proteins they encode in particular types/subtypes of MPS has been expanded. The new text is presented in following lines: 279, 298, 299, 302, 303, 309, 430-449, 451-458, 464,503-543. In addition, for clarity, Table 5 has been added, which contains information on the regulation of the expression of given genes in individual types/subtypes of MPS, as well as the role and localization of the products of these genes in the cell.

b) The details from microscopy should be clearly linked to protein and mucopolisacharides

RESPONSE:

The results of microscopic analyses are now linked to genes and their products and to MPS. The new text is presented in lines from 503-543 .

c) Also, additional information should be carefully revised and discussed about other neurological diseases and manifestations to avoid speculative sentences.

RESPONSE:

Discussion on other neurological diseases has been expanded, and new text is included in following lines: 423-424, 430-449, 453-456, 552-543.

d) Scope and limitations of the study should be clearly sentenced. As authors declared just prospective ideas regarding extension of application in other cells and patients.  

RESPONSE:

Scope and limitations of the study are now clearly indicated (lines: 583-587, 603-610).

e) Conclusions should be specific about the genes-proteins key in each MPS (some data are in the abstract, but no in the conclusions section). Some other -complementary- sentences should be at least in other paragraph.

RESPONSE:

We have followed the reviewer’s recommendations, and relevant changes have been included into Conclusions (lines: 583-587, 591-596, 603-610).

Reviewer 3 Report

Dear authors,

I have reviewed your manuscript titled "Cellular Organelle-related Transcriptomic Profile Abnormalities in Neuronopathic Types of Mucopolysaccharidosis: Comparison with Other Neurodegenerative Diseases" submitted for publication consideration in Current Issues in Molecular Biology. The study investigates gene expression changes related to cellular organelles in neuronopathic vs non-neuronopathic types of mucopolysaccharidosis (MPS), in order to identify candidate biomarkers and therapeutic targets for the neurological manifestations of MPS. This is an interesting and relevant research topic. While the study provides some useful findings, there are issues that need to be addressed before the manuscript can be accepted for publication.

Major revisions:

1. The rationale for using fibroblasts rather than neuronal cells for studying neurodegeneration needs to be explained and justified more clearly. What evidence is there that findings in fibroblasts can be extrapolated to neuronal abnormalities in MPS?

2. More details should be provided on the transcriptomic analysis methods and data quality control. How many biological replicates were used for RNA-seq? Were appropriate data normalization methods used?

3. The criteria for genes selected for further analyses seem quite arbitrary. Authors should provide statistical analyses to support the log2FC cutoffs used to identify genes with "severely impaired expression". Are these genes differentially expressed based on statistical testing?

4. Functional/mechanistic implications of the key genes with altered expression could be expanded and discussed further based on evidence from published literature. For example, are there reported neuronal phenotypes or CNS abnormalities associated with knockdowns of these genes? This would strengthen their potential relevance as neurodegeneration markers in MPS. 

Overall this an interesting study, but the above major points should be addressed to strengthen the results and conclusions drawn. I look forward to assessing a revised version that incorporates these revisions and provides more discussion of the functional/mechanistic relevance of these transcriptomic findings to MPS neuropathology. Please feel free to contact me if you have any questions regarding these comments and revision requirements.

Minor editing of English language required

Author Response

I have reviewed your manuscript titled "Cellular Organelle-related Transcriptomic Profile Abnormalities in Neuronopathic Types of Mucopolysaccharidosis: Comparison with Other Neurodegenerative Diseases" submitted for publication consideration in Current Issues in Molecular Biology. The study investigates gene expression changes related to cellular organelles in neuronopathic vs non-neuronopathic types of mucopolysaccharidosis (MPS), in order to identify candidate biomarkers and therapeutic targets for the neurological manifestations of MPS. This is an interesting and relevant research topic. While the study provides some useful findings, there are issues that need to be addressed before the manuscript can be accepted for publication.

Major revisions:

1. The rationale for using fibroblasts rather than neuronal cells for studying neurodegeneration needs to be explained and justified more clearly. What evidence is there that findings in fibroblasts can be extrapolated to neuronal abnormalities in MPS?

RESPONSE:

The rationale for the use of fibroblasts in studies on neurodegenerative diseases has been presented and discussed (lines: 561-580).

2. More details should be provided on the transcriptomic analysis methods and data quality control. How many biological replicates were used for RNA-seq? Were appropriate data normalization methods used?

RESPONSE:

Detailed transcriptomic method is described in the revised manuscript (lines: 143-157). Four biological repeats of each experiment were included, as indicated in lines 139-140.

3. The criteria for genes selected for further analyses seem quite arbitrary. Authors should provide statistical analyses to support the log2FC cutoffs used to identify genes with "severely impaired expression". Are these genes differentially expressed based on statistical testing?

RESPONSE:

The statistical methods used in analyses are described in the revised manuscript (lines: 159-166).

4. Functional/mechanistic implications of the key genes with altered expression could be expanded and discussed further based on evidence from published literature. For example, are there reported neuronal phenotypes or CNS abnormalities associated with knockdowns of these genes? This would strengthen their potential relevance as neurodegeneration markers in MPS. 

RESPONSE:

As requested by the reviewer, these aspects were discussed in more detail in the revised manuscript (lines: 423-424, 430-449, 453-456, 503-543).

Overall this an interesting study, but the above major points should be addressed to strengthen the results and conclusions drawn. I look forward to assessing a revised version that incorporates these revisions and provides more discussion of the functional/mechanistic relevance of these transcriptomic findings to MPS neuropathology. Please feel free to contact me if you have any questions regarding these comments and revision requirements.

RESPONSE:

We thank the reviewer for all the above points.  There were addressed in the revised manuscript and appropriate changes have been introduced.

Round 2

Reviewer 3 Report

Dear authors,

Thank you for submitting your revised manuscript titled "Cellular organelle-related transcriptomic profile abnormalities in neuronopathic types of mucopolysaccharidosis: comparison with other neurodegenerative diseases". I appreciate the efforts you have made to address the major points raised in the previous round of review. The additional information and analyses have strengthened the manuscript.

I have a few minor points that I believe would further improve the manuscript:

1. In the discussion on the rationale for using fibroblasts (lines 561-580), it would be helpful to briefly mention some key advantages of fibroblasts as a cellular model, such as their ease of obtainment from patients compared to neuronal cells. This would further justify the choice of fibroblasts.

2. The details provided on the transcriptomic methods (lines 143-157) are appreciated. Please also specify the number of reads and sequencing depth obtained per sample, as this information is relevant for assessing data quality and reliability of the differential expression results.

3. The expanded discussion on functional implications of the key differentially expressed genes (lines 423-424, 430-449, 453-456, 503-543) has improved the manuscript. I would suggest adding a couple of sentences on whether any of these genes have previously been implicated in lysosomal or neurodegenerative disorders, and if so, what functional roles they may play. This would help connect your findings to the broader context of these diseases.

4. Figure legends should be more detailed. For example, in Figure 1, please define in the legend what the asterisk (*) and hashtag (#) symbols denote in terms of statistical significance.

Overall, the revised manuscript has been improved and I believe it will be suitable for publication in Current Issues in Molecular Biology after these minor revisions are addressed. Please feel free to contact me if you have any questions.

Minor editing of English language required

Author Response

REVIEWER 3 – 2^nd review

Dear authors,

Thank you for submitting your revised manuscript titled "Cellular organelle-related transcriptomic profile abnormalities in neuronopathic types of mucopolysaccharidosis: comparison with other neurodegenerative diseases". I appreciate the efforts you have made to address the major points raised in the previous round of review. The additional information and analyses have strengthened the manuscript.

RESPONSE:

We are glad the reviewer is satisfied with the modifications introduced in the manuscript.

I have a few minor points that I believe would further improve the manuscript:

1. In the discussion on the rationale for using fibroblasts (lines 561-580), it would be helpful to briefly mention some key advantages of fibroblasts as a cellular model, such as their ease of obtainment from patients compared to neuronal cells. This would further justify the choice of fibroblasts.

RESPONSE:

We thank for this important suggestion. The arguments proposed by the reviewer have been added to the Discussion. The new test (lines 565-571) reads as follows:

“On the other hand, fibroblast could be relatively easily obtained from patients and con-trol individuals, making the research material both specific and homogeneous. The use of patient-derived cell lines is also advantageous relative to heavily modified cells, like neuronal cells differentiated under laboratory conditions, where potential off-target ef-fects should be taken into consideration, the problem which is absent in fibroblasts ob-tained directly from affected individuals.”

2. The details provided on the transcriptomic methods (lines 143-157) are appreciated. Please also specify the number of reads and sequencing depth obtained per sample, as this information is relevant for assessing data quality and reliability of the differential expression results.

RESPONSE:

The parameters requested by the reviewer are indicated in lines 145-147:

“The following sequencing parameters were obtained: PE150 (150 bp paired-end) and minimum 40 million (40 M) of raw reads, which gave a minimum of 12 Gb of raw data, per each sample.

3. The expanded discussion on functional implications of the key differentially expressed genes (lines 423-424, 430-449, 453-456, 503-543) has improved the manuscript. I would suggest adding a couple of sentences on whether any of these genes have previously been implicated in lysosomal or neurodegenerative disorders, and if so, what functional roles they may play. This would help connect your findings to the broader context of these diseases.

RESPONSE:

The diseases correlated to the discussed genes are indicated in Table 5 (the most right column).

4. Figure legends should be more detailed. For example, in Figure 1, please define in the legend what the asterisk (*) and hashtag (#) symbols denote in terms of statistical significance.

RESPONSE:

According to the reviewer’s request, the figure legends have been revised and missing explanations were added.

Overall, the revised manuscript has been improved and I believe it will be suitable for publication in Current Issues in Molecular Biology after these minor revisions are addressed. Please feel free to contact me if you have any questions.

             RESPONSE:

             As indicated above, all points raised by the reviewer were addressed.