Integrated Plasma and Glial Cell Evidence Indicates a Functional Role for hsa-miR-342-5p in Spinocerebellar Ataxia Type 7 and Its Potential Use as a Biomarker

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript investigates the potential of circulating microRNAs as prognostic biomarkers in spinocerebellar ataxia type 7 (SCA7), a rare neurodegenerative disorder with marked clinical heterogeneity. By analyzing the expression of selected neurodegeneration-associated miRNAs in plasma samples from SCA7 patients and in an inducible glial cell model, the authors aim to address an important unmet need for minimally invasive tools to improve disease diagnosis and monitoring. The study is timely and relevant, given the limited availability of biomarkers for SCA7, and it offers an integrated patient–cellular model approach to identify candidate miRNAs associated with disease severity and pathophysiology.

My comments are detailed below:

1. Throughout the manuscript, the authors use the term "prognosis" and "prognostic", while it seems that the biomarkers are "diagnostic" (differentiation between controls and SCA7). Prognosis refers to an outcome of a disease, once the disease was already diagnosed. Please change the wording accordingly, or else clarify why these markers are prognostic. 
2. Please mention the number of early onset (EO) patients and that of adult onset (AO) patients in table 1. 
3. The figure legends do not indicate what the symbols *, **, and *** represent in terms of statistical significance (e.g., p-value thresholds). This information should be added for clarity.
4. Figures 1-2 present the log 2 fold change in patients with individual points. How was the log2 fold change calculated for each patient? It will be better to present in these figures both the control group and the patient group with all individual points, for all miRNAs. I also suggest to add the titles of the panels above, rather than below the panels. 
5. In figures 3 and 4, "has" before the name of the miRNA should be changed to "hsa" (homo sapiens).
6. The authors show that EO patients show more robust changes in miRNA levels compared to controls, then AO patients. How were the levels of these miRNA changed in EO compared to AO?
7. Although the authors show significant differences between SCA7 patients and controls, it is not clear how well the miRNAs can distinguish between SCA7 and controls. Receiver operating characteristics (ROC) curves should be presented, with area under the curve (AUC), at least for some of the miRNAs. I also recommend to test the classification power of a combination of miRNAs. This could be analyzed with logistic regression. 
8. How are the levels of the miRNAs correlated with functional/clinical features of the disease or number of CAG repeats? if these data are available, it would be informative to perform this analysis. 
9.  The discussion should include mentioning of other biomarkers exisiting today for SCA7 (not necessarily miRNAs) and how the miRNAs identified perform with repsect to those markers. 

Minor comments:

1. line 36 - a word is missing between "patients" and "the".
2. line 105 - the terms "cohort validation analysis" in unclear. It seems that the cohort used here is not a validation cohort, but rather an initial cohort. 
3. The text in lines 404 - 411 repeats the text in lines 395-402 and should be removed.
4. line 427 - replace "=" with ">" as log2FC of 1 is a cut-off. 

Author Response

REVIEWER 1

This manuscript investigates the potential of circulating microRNAs as prognostic biomarkers in spinocerebellar ataxia type 7 (SCA7), a rare neurodegenerative disorder with marked clinical heterogeneity. By analyzing the expression of selected neurodegeneration-associated miRNAs in plasma samples from SCA7 patients and in an inducible glial cell model, the authors aim to address an important unmet need for minimally invasive tools to improve disease diagnosis and monitoring. The study is timely and relevant, given the limited availability of biomarkers for SCA7, and it offers an integrated patient–cellular model approach to identify candidate miRNAs associated with disease severity and pathophysiology.

My comments are detailed below:

1. Throughout the manuscript, the authors use the term "prognosis" and "prognostic", while it seems that the biomarkers are "diagnostic" (differentiation between controls and SCA7). Prognosis refers to an outcome of a disease, once the disease was already diagnosed. Please change the wording accordingly, or else clarify why these markers are prognostic. 

• We thank the reviewer for this comment. The four miRNAs selected in the present study were originally identified in our previous work as prognostic markers based on a comparative analysis between the Adult-Onset and Early-Onset phenotypes. Accordingly, in this study we aimed to validate these miRNAs in an independent cohort. In response to the reviewer’s suggestion, we have now clarified that these four miRNAs were selected from a prior study and had been proposed as potential prognostic markers; this information has been explicitly stated in the Materials and Methods section (4.3 miRNAs selection) and in the initial part of the Results section to avoid any confusion. In addition, in the figures we have softened the wording by describing them aspotential prognostic markers. Finally, we have performed a comparative analysis between the Adult-Onset and Early-Onset groups and a Receiver operating characteristic (ROC) curve analysis to assess potential differences between these two phenotypes, and the implications of these findings are showed in Results section and in the Discussion section.

2. Please mention the number of early onset (EO) patients and that of adult onset (AO) patients in table 1. 

• We have implemented this change in Table 1.

3. The figure legends do not indicate what the symbols *, **, and *** represent in terms of statistical significance (e.g., p-value thresholds). This information should be added for clarity.

• We have addressed this meaningful recommendation in the revised manuscript. We have now incorporated the levels of statistical significance into the figure legends to facilitate clearer interpretation for the reader.

4. Figures 1-2 present the log 2 fold change in patients with individual points. How was the log2 fold change calculated for each patient? It will be better to present in these figures both the control group and the patient group with all individual points, for all miRNAs. I also suggest to add the titles of the panels above, rather than below the panels. 

• We fully agree with this meaningful criticism. In the revised version of the manuscript, the calculation of log2 fold change (log2FC) is now explicitly described in the last paragraph of Section “4.5,Relative quantification of miRNAs”. We chose to use log2FC instead of fold change (FC) because this transformation provides a symmetric and statistically more robust representation of expression changes. Individual values from healthy controls are not shown because each sample was calibrated against a strictly age- and sex-matched control, which increases the statistical reliability of the comparisons. The use of log2FC allows equivalent interpretation of relative increases and decreases with respect to the calibrator (±1 corresponds to a twofold change), reduces the influence of extreme values, and stabilizes variance across paired samples. This approach facilitates consistent comparisons between individuals and minimizes biases derived from residual biological variability, thereby improving clarity and reproducibility. In addition, figures have been modified in accordance with the reviewer’s suggestion, and panel titles have been clearly highlighted above each graph.

5. In figures 3 and 4, "has" before the name of the miRNA should be changed to "hsa" (homo sapiens).

• The suggested changes were included in the Figure 3 and current figure 5 (previously figure 4).

6. The authors show that EO patients show more robust changes in miRNA levels compared to controls, then AO patients. How were the levels of these miRNA changed in EO compared to AO?

• We agree with the reviewer’s suggestion. In the revised version of the manuscript, we performed a direct comparison of the expression levels of the analyzed miRNAs between the adult-onset (AO) and early-onset (EO) groups. This additional analysis strengthens our original premise, as at least 2 miRNAs showed increased expression significantly in the more severe EO phenotype. We also explain the potential biases associated with comparing two heterogeneous groups; however, we link this limitation to the ROC curve analysis in order to provide greater robustness and confidence to the results. These results are now described in the fifth paragraph of the Results section entitled“1. Differential expression of circulating miRNAs in patients with SCA7” and we added a Supplementary Figure 1 with this analysis.

7. Although the authors show significant differences between SCA7 patients and controls, it is not clear how well the miRNAs can distinguish between SCA7 and controls. Receiver operating characteristics (ROC) curves should be presented, with area under the curve (AUC), at least for some of the miRNAs. I also recommend to test the classification power of a combination of miRNAs. This could be analyzed with logistic regression. 

• To approach this meaningful suggestion, we performed an additional prognostic analysis in the current cohort using the Mean expression of the twelve miRNAs associated with SCA7 severity (AO and EO) and the receiver operating characteristic (ROC) curves. This approach allowed us to identify a signature of twelve miRNAs analyzed that accurately discriminates SCA7 patients into adult-onset (AO) and early-onset (EO) groups. Moreover, ROC curve analysis demonstrated that this miRNA signature exhibits robust prognostic performance, with an area under the curve (AUC) of 0.799. These results have been incorporated into the revised manuscript and are described in the Results section (fifth paragraph of “1. Differential expression of circulating miRNAs in patients with SCA7”) as well as a current Figure 3.

8. How are the levels of the miRNAs correlated with functional/clinical features of the disease or number of CAG repeats? if these data are available, it would be informative to perform this analysis. 

• We are conscious that additional studies are needed to support the prognostic value of the SCA7-related miRNAs described in this study. As an attempt to reinforce our findings, we now described in the revised Manuscript the correlation between miRNAs with the CAG repeats number, age at onset and Factor S scale (see last paragraph of “1. Differential expression of circulating miRNAs in patients with SCA7”). Interestingly, several of these miRNAs showed significant correlations with CAG repeat length and age at onset. Specifically, hsa-miR-25-3p expression was significantly associated with both parameters in the overall SCA7 cohort, whereas hsa-miR-93-5p showed significant correlations in patients with the early-onset (EO) phenotype. In addition, other miRNAs were associated with CAG repeat length in the more severe phenotype, including hsa-miR-92a-3p and hsa-miR-106a-5p. All correlation analyses are provided in Supplementary Table 1. We hope that these additional results adequately address the reviewer’s comment.

9. The discussion should include mentioning of other biomarkers exisiting today for SCA7 (not necessarily miRNAs) and how the miRNAs identified perform with respect to those markers. 

• Following the reviewer’s valuable comment, we have added to the first paragraph of the Discussion the importance of identifying objective biomarkers for disease monitoring. Although SCA7 biomarkers have not yet been extensively characterized, we now briefly summarize those that have been evaluated to date and highlight the advantages of circulating miRNAs as objective biomarkers. We believe these additions improve the clarity of the study aims and provide a more solid framework for discussing our results.

Minor comments:

1. line 36 - a word is missing between "patients" and "the".

• We thank the reviewer for this valuable comment. The sentence has been corrected in the revised manuscript to improve clarity and avoid misinterpretation.

2. line 105 - the terms "cohort validation analysis" in unclear. It seems that the cohort used here is not a validation cohort, but rather an initial cohort. 

• We thank the reviewer for this appropriate comment and apologize for any confusion. Given that we had initial data from an SCA7 population, the validation cohort refers to a group of different patients selected from the SCA7 group, in whom independent experiments were performed to verify the presence of expression changes associated with the disease. We have now defined this cohort more precisely and changed the term “cohort validation” in the revised manuscript to avoid any misunderstanding and to provide greater clarity for the reader.

3. The text in lines 404 - 411 repeats the text in lines 395-402 and should be removed.

• This opportune observation was considered and then, the mentioned paragraphs were corrected.

4. line 427 - replace "=" with ">" as log2FC of 1 is a cut-off. 

• We have made the correction in accordance with the reviewer’s appropriate suggestion.

Reviewer 2 Report

Comments and Suggestions for Authors

In this study, the authors investigated circulating microRNAs in patients with the neurodegenerative disease spinocerebellar ataxia type 7 (SCA7), as well as microRNA profiles in a cellular model of the disease. The expression levels of 12 different microRNAs have been assessed, including previously proposed candidate prognostic biomarkers, and dysregulation in hsa-miR-342-5p expression levels has been demonstrated, both in patient samples and in the cellular model. A bioinformatics analysis has been performed to identify target genes for this microRNA. My feedback primarily concerns the manuscript’s formatting; however, I also have a few points that would benefit from clarification or additional detail in the text.

Comment 1. In the Introduction section, for example, in lines 48-50, 89-92, the same articles are cited repeatedly. Consider citing the sources only once to avoid redundancy.

Comment 2. Please provide additional detail on the molecular function of ataxin-7.

Comment 3. In lines 121 and 156, hyphens are missing in hsa-miR-483.

Comment 4. In Figure 2, line 152, please define the abbreviations "AO" and "EO" to allow readers to interpret the data independently of the main text.

Comment 5. In section 2.2, please specify that MIO-M1 cells were used.

Comment 6. Please explain why hsa-miR-483 expression in human samples was normalized to a single reference gene, while in the cell line experiments, multiple snoRNAs (U6, RNU44, and RNU48) were used for normalization. 

Comment 7. The captions for Figures 3 and 4 contain a typo in the microRNA name: “has-miR-…” should be corrected to “hsa-miR-…”

Comment 8. The sentence in lines 213-214 appears incomplete. Please revise for clarity.

Comment 9. In Figure 5, please move the sentence “The x-axis indicates the number of target genes per pathway, and the y-axis lists the corresponding KEGG pathways” to appear before point b).

Comment 10. Lines 258-260 state that miRNA dysregulation correlates with disease severity. However, no correlation analysis is presented in this study. To avoid overinterpretation, please consider softening the wording.

Comment 11. In lines 268 and 273, the terms in vitro and in vivo should be italicized.

Comment 12. Please ensure consistent formatting of “TGF-β” throughout the manuscript (e.g., in lines 217, 288, and 292, as well as in Figure 5).

Comment 13. The phrase “microRNA (miRNA)” appears in lines 307 and 311, even though “miRNA” has already been used throughout the manuscript.

Comment 14. Lines 322-323 state that this study is the first to characterize circulating microRNAs in SCA7. However, the same authors have previously reported on microRNA expression in SCA7 patients (10.1007/s12035-019-1480-y). To avoid overstatement, please revise this claim to accurately reflect the novelty of the current work.

Comment 15. Lines 341-347 contain several unnecessary line breaks that disrupt readability.

Comment 16. Please justify the unequal sex distribution in the cohort (25% women). Does this reflect the epidemiology of SCA7, or were other factors involved?

Comment 17. In lines 369 and 370, the temperature is written incorrectly as “°4C” and “4 C”. 

Comment 18. In lines 391 and 393, please correct the formatting of reference [45] and specify the duration of the incubation step at 85 °C.

Comment 19. In line 389, please clarify which reverse transcription kit was used.

Comment 20. The content in lines 404-411 repeats information from the previous section.

Comment 21. In lines 413-417, please specify the name of Thermo Fisher Scientific assays used to ensure clarity.

Comment 22. Line 420 states that 40 cycles were performed at 95 °C. This appears to be a misstatement,  please correct the description if needed. Was the same thermal cycling protocol, including annealing temperature, used for all microRNAs?

Comment 23. In line 433, “CO2” should be formatted with a subscript: CO₂.

Comment 24. Lines 456-460 state: “The comparison of miRNA expression in plasma between groups was determined using the non-parametric Mann Whitney U test.” Given that the choice of a non-parametric test implies the data were not normally distributed, please specify which statistical test or method was used to assess normality.

Comment 25. Among the miR-342-5p target genes used for KEGG analysis, please highlight some of those with experimental validation.

Author Response

REVIEWER 2

In this study, the authors investigated circulating microRNAs in patients with the neurodegenerative disease spinocerebellar ataxia type 7 (SCA7), as well as microRNA profiles in a cellular model of the disease. The expression levels of 12 different microRNAs have been assessed, including previously proposed candidate prognostic biomarkers, and dysregulation in hsa-miR-342-5p expression levels has been demonstrated, both in patient samples and in the cellular model. A bioinformatics analysis has been performed to identify target genes for this microRNA. My feedback primarily concerns the manuscript’s formatting; however, I also have a few points that would benefit from clarification or additional detail in the text.

Comment 1. In the Introduction section, for example, in lines 48-50, 89-92, the same articles are cited repeatedly. Consider citing the sources only once to avoid redundancy.

• This opportune observation was considered and then, the mentioned references were corrected.

Comment 2. Please provide additional detail on the molecular function of ataxin-7.

• We approached this meaningful recommendation in the revised manuscript. We now include a brief overview of the current understanding of ataxin-7 function, and highlight that its full range of biological roles has yet to be fully elucidated (see four paragraph of Introduction section).

Comment 3. In lines 121 and 156, hyphens are missing in hsa-miR-483.

• The suggested omission was mended.

Comment 4. In Figure 2, line 152, please define the abbreviations "AO" and "EO" to allow readers to interpret the data independently of the main text.

• Following the reviewer’s appropriate suggestion, the terms AO and EO have been defined in the legend of Figure 2.

Comment 5. In section 2.2, please specify that MIO-M1 cells were used.

• We now describe, in the “Results” section (first paragraph of the “2.2 Validation of plasmatic miRNAs in a human cellular model of SCA7 subsection), the characteristics of the MIO-M1 cells and their experimental contrasts.

Comment 6. Please explain why hsa-miR-483 expression in human samples was normalized to a single reference gene, while in the cell line experiments, multiple snoRNAs (U6, RNU44, and RNU48) were used for normalization. 

• We apologize for this omission. In the revised version of the manuscript, we now provide a detailed explanation of the selection of endogenous controls and their use for evaluating gene expression in patient samples, as well as, in the SCA7 experimental model (see the first paragraph of Section 4.5, “Relative quantification of miRNAs”).

Comment 7. The captions for Figures 3 and 4 contain a typo in the microRNA name: “has-miR-…” should be corrected to “hsa-miR-…”

• This mistake was corrected.

Comment 8. The sentence in lines 213-214 appears incomplete. Please revise for clarity.

• We thank the reviewer for this insightful comment. We have restructured the sentence to improve clarity and facilitate the reader’s understanding of the information presented.

Comment 9. In Figure 5, please move the sentence “The x-axis indicates the number of target genes per pathway, and the y-axis lists the corresponding KEGG pathways” to appear before point b).

• In accordance with the reviewer’s request, we have reorganized the legend of current Figure 6 (former figure 5)as suggested.

Comment 10. Lines 258-260 state that miRNA dysregulation correlates with disease severity. However, no correlation analysis is presented in this study. To avoid overinterpretation, please consider softening the wording.

• We thank the reviewer for this pertinent comment. In the revised version of the manuscript, we have softened the wording in suggested lines to avoid overinterpretation. In addition, we now include correlation analyses between miRNA expression changes and relevant clinical and molecular features, which provide further support and increase the robustness of our conclusions (see last paragraph of “1. Differential expression of circulating miRNAs in patients with SCA7”.

Comment 11. In lines 268 and 273, the terms in vitro and in vivo should be italicized.

• Following the reviewer’s comment, these terms are now italicized and used consistently throughout the manuscript.

Comment 12. Please ensure consistent formatting of “TGF-β” throughout the manuscript (e.g., in lines 217, 288, and 292, as well as in Figure 5).

• We thank the reviewer for this valuable suggestion, and the abbreviation has now been harmonized across the text.

Comment 13. The phrase “microRNA (miRNA)” appears in lines 307 and 311, even though “miRNA” has already been used throughout the manuscript.

• We appreciate the reviewer’s recommendation, and the “miRNA” concept has been consistently used throughout the manuscript.

Comment 14. Lines 322-323 state that this study is the first to characterize circulating microRNAs in SCA7. However, the same authors have previously reported on microRNA expression in SCA7 patients (10.1007/s12035-019-1480-y). To avoid overstatement, please revise this claim to accurately reflect the novelty of the current work.

• The reviewer's observation is very appropriate. We have modified the sentence to better reflect the originality of the present manuscript.

Comment 15. Lines 341-347 contain several unnecessary line breaks that disrupt readability.

• Thank you for your valuable feedback. We have carefully reviewed these lines and removed the unnecessary line breaks to enhance readability. This correction has been implemented in the revised manuscript. We appreciate your attention to detail in helping us improve our work.

Comment 16. Please justify the unequal sex distribution in the cohort (25% women). Does this reflect the epidemiology of SCA7, or were other factors involved?

• Indeed, from the evaluated cohort, the selected patients were those who met the predefined inclusion and exclusion criteria and who could be successfully matched with control subjects of the same age and sex from the same studied communities. In addition, given that greater genetic instability through paternal transmission has been well documented, the most severe cases (EO) in early generations are more frequently observed in males by patrilineal transmission. In response to the reviewer’s insightful comment, the current version of the manuscript briefly discusses the underlying cause of this difference and its potential implications for the results obtained. We also suggest that future studies should evaluate a larger cohort including both sexes to determine whether sex-related differences are present (see second paragraph of discussion).

Comment 17. In lines 369 and 370, the temperature is written incorrectly as “°4C” and “4 C”. 

• The correction was mended

Comment 18. In lines 391 and 393, please correct the formatting of reference [45] and specify the duration of the incubation step at 85 °C.

• Thank you for your feedback. We have corrected the formatting of reference [45] as requested and specified the duration of the incubation step at 85 °C in the revised manuscript. We appreciate your attention to these details.

Comment 19. In line 389, please clarify which reverse transcription kit was used.

• Thank you for this comment. We have now clarified that the reverse transcription was performed using the (Taqman ^TM MicroRNA Reverse Transcription Kit; Thermo Fisher Scientific, Walthman, MA, USA), and this information has been added to the Materials and Methods section for clarity and reproducibility.

Comment 20. The content in lines 404-411 repeats information from the previous section.

• This opportune observation was considered and then, the mentioned paragraphs were corrected; in the current version of the manuscript, the information is no longer duplicated.

Comment 21. In lines 413-417, please specify the name of Thermo Fisher Scientific assays used to ensure clarity.

• This information was added (The identification numbers for each assay have been incorporated into the text).

Comment 22. Line 420 states that 40 cycles were performed at 95 °C. This appears to be a misstatement,  please correct the description if needed. Was the same thermal cycling protocol, including annealing temperature, used for all microRNAs.

• The complete protocol information has been added to the second paragraph of Section 4.5. , “Relative quantification of miRNAs”.

Comment 23. In line 433, “CO2” should be formatted with a subscript: CO₂.

• The correction was mended

Comment 24. Lines 456-460 state: “The comparison of miRNA expression in plasma between groups was determined using the non-parametric Mann Whitney U test.” Given that the choice of a non-parametric test implies the data were not normally distributed, please specify which statistical test or method was used to assess normality.

• Thank you very much for this insightful comment. In accordance with the reviewer’s observation, we have now specified the statistical test used to assess data normality (Shapiro-Wilk test,) and clarified the rationale for using the non-parametric Mann–Whitney U test. This information has been added to the second paragraph of Section“8. Statistical Analysis”.

Comment 25. Among the miR-342-5p target genes used for KEGG analysis, please highlight some of those with experimental validation.

• We have addressed this meaningful recommendation in the revised manuscript. We now include a brief discussion, supported by experimental evidence, of several miR-342 target genes that have been identified in transcriptional analyses related to SCA7, such as BMP7, CCND1, Raf1 and CFL1 (see fifth paragraph of Discussion section).

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have provided sufficient answers to my questions and have amended their manuscript accordingly. The revised manuscript can be accepted after text editing.