Review Reports

Response to Reviewer 1 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

Is the research design appropriate?

Yes

Are the methods adequately described?

Yes

Are the results clearly presented?

Yes

Are the conclusions supported by the results?

Yes

Are all figures and tables clear and well-presented?

Yes

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: [This research examined the roles of oncomodulin (OCM) and cadherin 23 (CDH23) in age-related hearing loss (AHL), focusing on their influence on outer hair cell (OHC) function and susceptibility to ototoxic stress. The author introduced a new Ocm-knockout mouse model (Ocmtm1a/tm1a) to assess the effects of OCM deficiency alongside the Cdh23753G→A mutation, which is associated with early-onset AHL. The results showed that Ocm-KO mice with the Cdh23753G→A mutation experienced significant hearing loss and a reduction in OHCs by the age of three months. Conversely, Ocm-KO mice without this specific Cdh23 mutation did not show similar hearing loss until they were 12 months old, indicating that the Cdh23 mutation worsens early age-related hearing loss in OCM-deficient mice. Another part of the study explored OCM's protective role against ototoxic stress caused by 2-hydroxypropyl-β-cyclodextrin (HPBCD). Ocm-KO mice exhibited a more severe decline in OHC function, along with greater structural damage and faster rupture events in OHCs, when exposed to HPBCD, both in vivo and in vitro. The study findings provide important insights into the molecular and audiological aspects of hearing and the mechanisms of ototoxicity. I thoroughly enjoyed reading this outstanding research article. I did not identify any factual errors or major inconsistencies that required correction in the paper. The article is well-organized, and the language is clear. The quality of the research is exceptionally high.]

Response 1: Thank you.

4. Response to Comments on the Quality of English Language

Point 1: [The English could be improved to more clearly express the research.]

Response 1: Thank you for pointing this out. We, including a native speaker, have carefully double-checked the English.

Response to Reviewer 2 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Can be improved

Is the research design appropriate?

Yes

Are the methods adequately described?

Can be improved

Are the results clearly presented?

Yes

Are the conclusions supported by the results?

Can be improved

Are all figures and tables clear and well-presented?

Yes

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: [The manuscript addresses an important question regarding the interaction between Ca²⁺ buffering and genetic susceptibility in age-related hearing loss. The use of a novel Ocm-KO model combined with functional and structural analyses represents a clear strength.

However, several aspects require clarification and improvement. The interpretation that the Cdh23 mutation is solely responsible for accelerated hearing loss should be moderated, as residual genetic modifiers cannot be excluded ("suggests a major contribution” instead of “solely responsible).]

Response 1: We removed the “solely” from abstract and conclusion. We have revised a sentence that “Our data (Figures 2-4) suggest that a Cdh23^753G→A mutation, rather than other possible strain-specific genetic contributors to AHL, is solely responsible for exacerbating the AHL phenotypes of Ocm-KO mice.” to “Our data (Figures 2-4) suggest that a Cdh23^753G→A mutation is a major contributor to exacerbating the AHL phenotypes of Ocm-KO mice.” in Lines 365-368 of the revised manuscript.

Comments 2: [Additionally, the relatively small sample sizes in several experiments limit statistical robustness and should be acknowledged as a limitation.]

Response 2: We have added the following sentences to the Statistics section of the Materials and Methods in Lines 524-534 of the revised manuscript:

To determine the required sample sizes, a priori power analyses were conducted using G*Power [83]. For DPOAE thresholds, OHC loss with age, and OHC survival under in vitro HPβCD treatment, we selected the ANOVA: Fixed effects, special, main effects and interactions test (effect size = 0.46-0.84, alpha = 0.05, power = 0.8, number of groups = 6-8), indicating sample sizes of 3, 3, and 7 per group, respectively. For OHC loss under in vivo HPβCD treatment, we used the ANOVA: Fixed effects, omnibus, one-way test (effect size = 1.3, alpha = 0.05, power = 0.8, number of groups = 4), indicating a sample size of 3 per group. For relative mRNA levels of Ocm, the Means: Difference between two independent means (two groups) test was used (effect size = 3.1, alpha = 0.05, power = 0.8), resulting in a sample size of 3 per group. Effect sizes were determined based on our previous publications [37, 38, 40, 73, 84] and preliminary data.

Comments 3: [The reliance on DPOAE as the only functional measure restricts conclusions to outer hair cell function. The absence of ABR recordings should be explicitly discussed. Clarify that conclusions are limited to OHC function.]

Response 3: In the adult cochlea, OCM is predominantly expressed in OHCs. The lack of OCM leads to progressive hearing loss, as demonstrated by DPOAE and ABR in previous Ocm-KO mouse models. As such, we focus on DPOAE measurement. Nevertheless, we have added ABR data to the revised Supplementary Figure 1 and the associated Figure Legend in the revised Supplementary Materials. The Results in Lines 199-201 and Materials and Methods in Lines 478-490 of the revised manuscript have been modified accordingly.

Comments 4: [Furthermore, the HPβCD model, while useful, represents an extreme ototoxic insult, and its translational relevance to age-related hearing loss should be addressed.]

Response 4: We agree that our HPβCD model is an extreme, high dose model. It is true that this model is not designed to mimic the gradual progression of age-related hearing loss, but rather to create a rapid onset of hearing loss and OHC damage.

We have added “high dose” to the HPβCD treatment in vivo section of the Materials and Methods in Line 495 of the revised manuscript.

We have also added the following sentence in Lines 496-497 of the revised manuscript:

A major adverse effect of using high doses of HPβCD to treat Niemann-Pick disease type C is hearing loss [29, 31].

Comments 5: [Overall, the manuscript is promising but would benefit from a more balanced discussion (there is overinterpretation in some parts and needs clearer distinction: what is demonstrated vs. what is hypothesized).]

Response 5: Thanks for the suggestions. We have removed over interpretation that was not demonstrated, and focused on the data in the paper.   

Response to Reviewer 3 Comments

1. Summary

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted in the re-submitted files.

2. Questions for General Evaluation

Reviewer’s Evaluation

Response and Revisions

Does the introduction provide sufficient background and include all relevant references?

Yes

Is the research design appropriate?

Must be improved

Are the methods adequately described?

Must be improved

Are the results clearly presented?

Must be improved

Are the conclusions supported by the results?

Must be improved

Are all figures and tables clear and well-presented?

Yes

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: [It is an interesting piece of work, but it needs substantial improvement. I will present my pointys step bby step:

1. Introduction==> This part of the text needs substantial improvement. Thye main aspects of the  the theory (substances, molecules , point and aims of the study) must be re-written.  The purposes shall be clearly reported]

Response 1: 

In the first paragraph, we introduce the cochlear organ, the outer hair cells, and the OCM molecule, highlighting their roles in the process of hearing. 

In the second paragraph, we discuss the current knowledge gap related to OCM. 

The third paragraph outlines our study's objectives and strategy. Specifically, the purposes of the study and the substances/molecules used are reported in the Introduction in Lines 81-97 of the revised manuscript.

Comments 2: [2. Materials and Methods==-> The whole part is missing!!! Thye authors must explaine why they have used this kind of animals.]

Response 2: 

We used more than 1500 words to describe the Materials and Methods in Lines 400-535.

In the Introduction, we used two paragraphs to explain what the knowledge gap in the literature is (Line 65-81) and why we created this mouse line (Line 82-85). The detail of how we created this mouse line is further explained in the Animals section of the Materials and Methods in Lines 409-418 of the revised manuscript.

Comments 3: [They just also present a statistical method on the amount of the animals used and studied (how many, why, are they enough for the parameters studied?).]

Response 3: We have added the following sentences to the Statistics section of the Materials and Methods in Lines 524-534 of the revised manuscript:

To determine the required sample sizes, a priori power analyses were conducted using G*Power [83]. For DPOAE thresholds, OHC loss with age, and OHC survival under in vitro HPβCD treatment, we selected the ANOVA: Fixed effects, special, main effects and interactions test (effect size = 0.46-0.84, alpha = 0.05, power = 0.8, number of groups = 6-8), indicating sample sizes of 3, 3, and 7 per group, respectively. For OHC loss under in vivo HPβCD treatment, we used the ANOVA: Fixed effects, omnibus, one-way test (effect size = 1.3, alpha = 0.05, power = 0.8, number of groups = 4), indicating a sample size of 3 per group. For relative mRNA levels of Ocm, the Means: Difference between two independent means (two groups) test was used (effect size = 3.1, alpha = 0.05, power = 0.8), resulting in a sample size of 3 per group. Effect sizes were determined based on our previous publications [37, 38, 40, 73, 84] and preliminary data.

Comments 4: [3. 3. Results==> As far as the amount of the animals is not clear, no reliable results can presented and accepted. More diagramms would be halpful.]

Response 4: The numbers of animals used for each experiment were in Figure Legends in Lines 126-127, 163, 175-176, 181-182, 235-236, 267-268, 296-297, 336 and the associated Figures.

Comments 5: [4. Discussion==> The discussion shall bhe written based on thye findings. In my opinion, as far as we don't have reliable results, the discussion is incomplete.]

Response 5: Our findings are described and discussed in the Discussion in Lines 340-345, 365-368 of the revised manuscript. The conclusion is summarized in Lines 395-399.

Comments 6: [Legends of the figures==> Thyey must be more descriptive.]

Response 6: We have double-checked the details in the Figure Legends. We also added conclusions for each figure.