MANF supports the inner hair cell synapse and the outer hair cell stereocilia bundle in the cochlea

The authors show in the mouse how the auditory hair cell structural maintenance is perturbed by the inactivation of Manf and the concomitant ER stress, causing early-onset, progressive hearing loss.

2. The work done is detailed and extensive, with support of the hypotheses presented.
3. Additional issues to be addressed Line 42 We propose the involvement of this mechanism in the pathophysiology of Usher syndromes. There is no evidence that suggests this mechanism is involved in US, which is a combination of deafness and RP. The only phenotype described in the manuscript is deafness. Please remove and replace "Usher syndromes" with "deafness." While Cdh23 is associated with Usher syndrome, there is no additional evidence regarding this syndrome, rather than deafness alone.

Line 189
Remove 'data not shown', Either show the data in supplementary or take out this sentence. It is no longer acceptable to make this statement.
I am unable to access Montaser et al paper without payment; therefore, it is hard to judge if the data on the human patient is novel, or had already been described in the previous manuscript.
Reviewer #2 (Comments to the Authors (Required)): The study by Ikäheimo et al investigates how endoplasmic reticulum (ER) stress elicits hair cell pathology, using mouse models with inactivation of Manf (Mesencephalic astrocyte-derived neurotrophic factor). MANF is an endoplasmic reticulum protein that promotes ER homeostasis and has a cytoprotective function. When Manf was conditionally knocked out in Pax2 cre mice this caused hearing loss from early stages that was likely to result from abnormal OHCs. The OHCs had disorganised stereocilia, being worse affected in the basal coil. The hearing loss progressed as did the OHC stereocilia deformity, showing fusion of stereocilia and OHC death. By contrast the IHCs had normal bundles but lost up to 50% of ribbon synapses in the basal coil. All of these abnormalities were correlated with genetic background, only being present in mice with a cdh23 mutation. This was assumed to be due to the extra ER stress in these animals that is pushed over the limit by manf dysfunction. Finally they show a human with similar deafness due to mutation in manf. The ms is well written and the experiments are performed well. I just have a few main questions.
1. The previous publication from the lab is very similar to this study with a lot of overlap. I understand that the mice have been investigated at an earlier stage but I think the main messages of the papers remain very similar. The study is fairly descriptive and does not offer any mechanistic advance on what is already established.
2. What cells does the pax2 cre target? There is no explanation or characterisation of manf expression or lack of it.
3. Are the effects of manf KO really related to a direct effect of manf or is it a developmental indirect effect? For instance, could it be something developmental related to mutation in cdh23? This needs to at least be clearly explained in the ms i.e. that all the effects could be secondary to some developmental defect.
4. The mouse genetic background of the KO seems quite a mess. Five or six generations are not enough to get pure breed, at least 10 generations are required. This should be explicitly explained.
5. The controls should also be cre positive with non-mutant manf. This would ensure that the Pax2 cre expression is not having any direct effect.
6. Have the afferents and ribbon synapses been characterised in the OHCs? This would be a good comparison to make with the IHCs. Are the mechanisms affecting the synapses in both hair cell types similar or not? 7. The information on the human study is very limited. There is nothing mentioned of any other underlying defect or mutation that could bring the manf mutation into effect. The author's show that the manf KO has no affect by itself so what is going on in this patient?
8. The IHC controls in figure 3 should be shown since the expression patterns seem to be different to the OHCs. This should also be explained. figure 5N why is only one mouse used? Why not use both mice for this? The numbers of mice are very low anyway at n=2 for the analysis of TEM ribbon morphology so at least use all you have. Were the ribbons in CD-1 mice investigated at all? It would be interesting to see if the numbers are comparably low, although I understand there is no control for these.

In
10. If you are drawing conclusions from data then they should be shown -line 173-179 11. The figures should be labeled in a way that would make them easier to read. Write what the colors of the staining represent, label what is in the picture i.e. WT or cKO etc. Also label the axes on the human hearing data.
Reviewer #3 (Comments to the Authors (Required)): In this study, the authors expand on their previous finding that mice deficient in Manf, an ER stress mediator, develop hearing loss. They show evidence of stereociliary disruption and, ultimately, fusion in outer hair cells as well as loss of loss of ribbon synapses in inner hair cells. They demonstrate that this phenotype is likely dependent on the presence of Cdh23 Ahl allele, which is strongly associated with rapid age-related hearing loss due to disruption of stereociliary tip links. Finally, they provide evidence from a single human subject that Manf deficiency is related to hearing loss in humans. This study provides important corroboration for the role of Manf (and, by extension, ER stress) in hearing loss.
However, one critical timing problem limits the impact of their conclusion, Specifically, they show that Manf cKO mice have significant hearing loss, particularly at high frequencies, as early as P15 (shortly after the onset of hearing). In fact, the highfrequency thresholds are largely the same at P15 and P56. However, they find evidence of subtle stereociliary splaying in both high-and low-frequency outer hair cells at P22 (though they do not show the images for the low-frequency OHCs), and only evidence of sterociliary fusion at P56. Therefore, the time course and frequency specificity of their stereociliary phenotype (splaying and/or fusion) does not match with the timing of the hearing phenotype. This calls into question any conclusion they may draw into the specific mechanistic role through which Manf deficiency causes hearing loss through structural changes in the stereocilia, which is a central point of the paper.
The other points of the paper -the apparent synergy of the Ahl allele and Manf-induced hearing loss; and the case report of the human with Manf variant and hearing loss -are interesting. Though the Ahl/Manf finding should be discussed in contrast with the previous finding from Li et al (JCI, 2018) that TMTC4, another ER-stress-associated hearing-loss gene, still had a rapid progressive hearing loss phenotype even when outcrossed from B6 to FVB, suggesting that the Cdh23 Ahl allele is not required for ER-stress-associated hearing loss.
Overall, the authors provide a very technically strong, detailed characterization of the stereociliary changes that occur in their Manf cKO. However, all of this detail does not necessarily provide meaningful physiological insight into the mechanism by which Manf essentially is potentiating the already-known effect of the Cdh23 Ahl allele, and is therefore of somewhat limited impact.

Appeal Request
May 26, 2021 *Re: Life Science Alliance Manuscript -Editorial Decision LSA-2021-01068-T Dear Editor-in-Chief, Thank you very much for getting back to us. We are of course disappointed by your decision not to allow us to prepare a revised ms. The comments by reviewers were constructive and we can address their criticism. We do not agree with the comment of extensive similarity with our previous paper. Therefore, we would be grateful if you reconsider your decision, based on our comments below.
-----------*Editor comment #1:* The advance of this manuscript over your previous publication, Herranen et al, 2020, remains very limited. While confirmatory studies are acceptable at LSA, we agree with the reviewer that the two publications are very similar with a lot of overlap in data and takehome message.
*Author response:* The criticism of similarity between the ms and our published paper was raised by *one out of three reviewers*. In the published paper, we showed in adult mice that *Manf* inactivation leads to hearing loss and that it is associated with hair cell death. While it is true that hair cell death is a part of the mutant phenotype, the current ms shows that *non-functionality of hair cells *is the primary reason for the severe hearing deficit and that the non-functionality is present already at the age when hearing function commences. We show *novel molecular data of how ER stress perturbs the integrity of the mechanotransduction domain (hair bundle) and of the hair cell synapse (ribbon synapse),* these being separate events from the activation of proapoptotic pathways. Of course, because the same mutant mouse model was analyzed, the final manifestation of the mutant phenotype-hearing loss and hair cell death-remains the same in the ms and the published paper. In all, *the ms provides new insight of the molecular events and genetic status that are associated to the hearing loss phenotype. * *-----------* *Editor comment #2*: Reviewer 2 points out that the Manf-KO phenotypes could be secondary to a defect in embryonic development in general, and that Manf-KO mice have not been bred for enough numbers of generations (at least 10 would be required) to make it a pure-bred. *Editor comment #3*: Since the paper is a mechanistic follow-up of the previous study, the point made by Reviewer 3 that all of this detail does not necessarily provide meaningful physiological insight into the mechanism by which Manf essentially is potentiating the already-known effect of the Cdh23 Ahl allele is also of concern. *Author response:* Reviewer 2 did not suggest a possible contribution of *Manf* inactivation in *embryonic* development, rather pointed out Manf's putative developmental (maturation) role after birth. Reviewer 2 is right in writing that fine-grained development of stereocilia could be hampered in the mutant mice. This development could include the formation of stereocilia links required for hair bundle cohesion (lateral links) and mechanotransduction (tip links) and, thus, for the onset of hearing function. This topic is related to the criticism by reviewer 3 "the ms lacks physiological insight how Manf potentiates the effect of the mutant Cdh23 allele, because Cdh23 is a component of these links". This is the *main criticism raised by the reviewers. We think that it is feasible to address the criticism* by new experiments. Therefore, we ask you to reconsider your decision. In the revised ms, we would provide evidence that stereocilia links are defective in *Manf* mutant mice under the C57BL6 background. Cdh23 is known to be a component of the stereocilia links and the C57BL6 background is known to carry the mutant *Cdh23* allele. Thus, we think that "adding insult to insult", i.e. ER stress due to *Manf* inactivation combined with ER stress due to* Cdh23* mutation (Blanco-Sánchez et al. 2014), impairs the development or maintenance of the stereocilia links, causing early-onset hearing loss. Along these lines, we can address the other criticism by reviewer 2 "the time course and frequency specificity of their stereociliary phenotype (splaying and/or fusion) does not match with the timing of the hearing phenotype". We would provide evidence of a tonotopic (high-to-low-frequency) gradient along the cochlea in the presence of the stereocilia links, a fact that could explain the prominent elevation of hearing thresholds specifically in the high frequencies and already at the onset of hearing. The argument that *Manf* KO mice have not been bred for enough numbers of generations to make it a pure-bred is not relevant for the data presented because we confirmed by sequencing the status of the *Cdh23* allele, i.e. we know that the animals analyzed are homozygous for the mutant allele. ------------ The reviewers are inner ear experts with excellent comments. Reviewer 1: "Multiple mechanisms are associated with normal function of the inner ear, and pathogenesis leading to deafness. One of these, which is understudied, is the role of the endoplasmic reticulum (ER) in hearing and ER stress associated with deafness. Given the sensitivity of hair bundles and ribbon synapses to stress, and the ubiquity of ER stress in many diseases' pathogenesis, this is a particular compelling area to study. The work provides an advance in this area, outlining in detail the effects of ER stress and contribution to hair cell damage and the pathophysiology of deafness". The other reviewer commented that "the ms is technically strong". Based on these supportive comments and the fact that the criticism raised is not extensive and is possible to address, we cordially ask for reconsideration allowing us to revise the ms.
We strongly believe on the impact of our data to the auditory field and more broadly to the cell biological research on ER stress, and we think that your journal would be an appropriate forum for these data. We are looking forward to hearing from you soon. We are pleased to share with you that if you are able to address the Editor comment 2 and 3 with additional data, as you have laid out in your email below, we would be happy to re-consider the manuscript at LSA.
We would advise you to submit an appeal with a pbp rebuttal and a revised manuscript (that includes the additional data) when you are ready.
Please let us know if you have any further questions.

REBUTTAL TO THE COMMENTS RAISED BY THE REFEREES
We thank the editors and the referees for the many constructive comments and suggestions. We have carried out extensive new experimentation to address the concerns and we have made necessary additions and changes to the text and figure material.
Editor #1, The advance of this manuscript over your previous publication, Herranen et al, 2020, remains very limited. While confirmatory studies are acceptable at LSA, we agree with the reviewer that the two publications are very similar with a lot of overlap in data and takehome message.
Response #1: We will first address the criticism that the current ms is very similar with our previous publication (Herranen et al., 2020). This criticism was raised by one out of the three referees. In both cases, the same mutant mouse model (Manf cKO mice) has been studied to understand the importance of the maintenance of ER homeostasis in the cochlear cells and in hearing function. Should one consider only the final functional phenotype, the end result is the same: severe sensorineural hearing loss. In the previous paper, we showed that the hearing loss at adulthood was accompanied by ABR threshold elevations and robust hair cell loss and we did not conclude anything more precise about the underlying cellular events nor the temporal progression of the hearing loss. Now that we have continued this research, we found that the causes of the hearing loss are more fundamental and are initiated much earlier. This allowed us to delve deeper into the topic. To add, our finding of an early-onset hearing loss phenotype in a MANF-variant patient brings more interspecies relevance to our analysis of young Manf-inactivated hair cells.
In the current ms, we show that the disruption of the critically important functional domains of hair cells, the hair bundle and ribbon synapses, is the primary cause of hearing loss in Manf cKO mice. We show that the hair bundle impairment is present already when hearing function starts at the juvenile life. We deal with the mechanisms that drive the progressive hair bundle pathophysiology. These novel findings show how the ER stress related pathology affects cell function long before inducing activation of the pro-apoptotic pathways. We think that this is important for a broader audience; in most cases pathological ER stress is studied in the context of cell death, while the current manuscript show its importance in driving dysfunction of cells.
Here, we specify the novel findings of the current manuscript: (1) The OHC hair bundles show abnormal structure from the hearing onset, which corroborates the ABR results at this stage and present the plausible proximal cause of the DPOAE threshold elevations.
(2) We demonstrate a novel molecular mechanism of how the fusion of stereocilia in the OHC hair bundle proceeds. Stereocilia fusion has been described previously in pathological contexts, but here, we are able to deepen the understanding of the underlying mechanisms. (3) We show that IHC synaptopathy has an early onset and that it involves the loss of both pre-and postsynaptic structures, indicating non-functionality of the synapse. We deepen the knowledge of the spatial pattern of ribbon synapse loss across the modiolar-pillar axis of IHCs, important since it is known that noise-induced synaptopathy has a spatial preference linked to the functional characteristics of the synapses. We add data on the ultrastructure of the ribbon synapse by showing morphological abnormalities in the synaptopathic IHCs. (4) We show that Manf inactivation does not abolish neuroplastin expression in OHC hair bundles.
Recent data show that MANF can bind neuroplastin, and neuroplastin has been suggested to be the long-sought receptor for MANF. This is important considering other recent data that neuroplastin is required for PMCA2 expression in OHC stereocilia. However, we show that the stereocilia fusion event leads to neuroplastin and PMCA2 downregulation and thereby degraded Ca 2+ clearance. (5) We show novel human audiological data by analysing a loss-of-function MANF patient.
In all, in the published paper we revealed the role of MANF as a survival factor. In the present paper, we demonstrate the important contribution of Manf (ER homeostasis) to the maintenance of hair cell physiology and function, independently of cell death.
Editor #2, Reviewer 2 points out that the Manf-KO phenotypes could be secondary to a defect in embryonic development in general, and that Manf-KO mice have not been bred for enough numbers of generations (at least 10 would be required) to make it a pure-bred Response #2: In the original ms., we showed that all hair cells (P22) and ribbon synapses are formed (P22) in cKO cochleas. We have added to revised ms data on the OHC hair bundle status in the immature (P9) mutant mice. We do not see defects at the level of hair bundles, for example bundle abnormalities seen in Usher mutant mouse models. We have addressed the question of the contribution of developmental abnormalities to the altered bundle phenotype in our response below to referee#2 and dealt with this topic in the revised ms (results lines 121-127; discussion lines 350-363, revised ms Figs 3 and S2). While we have not bred the CBA/Ca KO mouse line to a pure bred, we have confirmed that all individual analysed lacked the ahl variant of Cadherin-23. Thus, we think that the requirement of a pure bred is not relevant. The data are summarized in Table 1.
Editor #3, Since the paper is a mechanistic follow-up of the previous study, the point made by Reviewer 3 that all of this detail does not necessarily provide meaningful physiological insight into the mechanism by which Manf essentially is potentiating the already-known effect of the Cdh23 Ahl allele is also of concern.
Response #3: We thoroughly agree with the comments of referee#3. They are essentially same as those by referee#2. We specify our conclusions in our responses below to referees#2 and #3. The topic has been discussed in revised ms (discussion lines 350-363).
Here we list the experiments made during the revision process to address this topic: (1) We studied the pre-hearing developmental aspects of OHC stereocilia to elucidate whether Manf deficiency could potentiate some early deficit that the Cdh23 ahl missense mutation alone does not trigger. We were able to find that the OHC hair bundles of Manf cKO B6 (P9) mice are structurally comparable to age-matched control B6 mice. We could find that both genotypes develop a complement of inter-stereociliary links, many of which are known to contain Cdh23. We could not directly show if and how Manf inactivation (ER stress) plays together with the Cdh23 ahl mutation. We would like to point out that very little is understood of the molecular/cell biological mechanism how Cdh23 ahl mutation itself leads to progressive hearing loss typical to the B6 mouse strain. (2) In our attempts to reveal the mechanisms of the early-onset hearing loss of Manf cKO mice, we studied the possibility that PMCA2-dependent Ca 2+ clearance is impaired in OHC stereocilia. This was an attractive possibility since Manf has been recently show to be able to bind neuroplastin in cell culture. Another recent study nicely demonstrated the tight association between neuroplastin and PMCA2 in OHC stereocilia (both papers cited in revised ms). We did not find Manf to be indispensable for the interaction of neuroplastin with PMCA2. However, we show in revised ms that the end result of the OHC pathology in adult Manf cKO mice, stereocilia fusion, is associated with downregulation of neuroplastin and PMCA2 and, therefore, with impaired Ca 2+ dynamics. While it is speculative to assume that stereocilia fusion proceeds always the same way (even without Manf inactivation or ER stress), this can be useful knowledge when it comes to studying the mechanisms underlying the loss of OHC function or OHC death.

Reviewer #1 (Comments to the Authors (Required)):
Line 42. We propose the involvement of this mechanism in the pathophysiology of Usher syndromes. There is no evidence that suggests this mechanism is involved in US, which is a combination of deafness and RP. The only phenotype described in the manuscript is deafness. Please remove and replace "Usher syndromes" with "deafness." While Cdh23 is associated with Usher syndrome, there is no additional evidence regarding this syndrome, rather than deafness alone.
Response: This last sentence of the abstract has been omitted from the revised ms.

Reviewer #2 (Comments to the Authors (Required)):
1. The previous publication from the lab is very similar to this study with a lot of overlap. I understand that the mice have been investigated at an earlier stage but I think the main messages of the papers remain very similar. The study is fairly descriptive and does not offer any mechanistic advance on what is already established Response: We disagree with this comment, but have taken it very seriously. It is our major mistake that we did not clearly express the novel messages in original ms. Above as a response to Editor's comment#1, we specify in detail the new findings of our ms, including the novel data in revised ms.
2. What cells does the pax2 cre target? There is no explanation or characterisation of manf expression or lack of it. Response: Pax2 is expressed from the early-embryonic stages onward in the inner ear; in the otic epithelial cells and in spiral and vestibular ganglion sensory neurons (Ohyama and Groves, 2004 (Blanco-Sanchez et al., 2014).
4. The mouse genetic background of the KO seems quite a mess. Five or six generations are not enough to get pure breed, at least 10 generations are required. This should be explicitly explained. Comment: (Also response Editor#2 comment). To be able to perform this study in a decent time constraint (not waiting for a few years to get the pure bred Manf KO CBA line), we have sequenced all our CBA individuals for the Cdh23 gene (wild type/mutant heterozygous/mutant homozygous). Table 1 summarized these data.
5. The controls should also be cre positive with non-mutant manf. This would ensure that the Pax2 cre expression is not having any direct effect. Comment: As controls, we have used wild types, fl/+ and Cre+ mice. We have now changed this throughout the revised ms, so that we compare mutants to controls and we define the control status (materials and methods, line 672). Just to mention that we used in a recent paper the same Cre-line for conditional gene (Mdm2) inactivation in the inner ear (Laos et al. Sci Rep 2017).
6. Have the afferents and ribbon synapses been characterised in the OHCs? This would be a good comparison to make with the IHCs. Are the mechanisms affecting the synapses in both hair cell types similar or not? Comment: In revised ms, we show a comparable OHC ribbon status in Manf cKO and control mice (revised ms Fig. S5, CtBP2 data). We have also studies these synapses using TEM, but the amount of OHC ribbons is too small to put it to a publication. This is an interesting topic. We do not have any answer to the IHC-OHC ribbon synapse difference. Is the ER differently located in the basal pole of IHCs and OHCs? Are there differences in Ca 2+ buffering capacity at these locations, considering that ER stress is linked with ER-Ca2+ depletion? Etc. This is too speculative to add to the discussion.
7. The information on the human study is very limited. There is nothing mentioned of any other underlying defect or mutation that could bring the manf mutation into effect. The author's show that the manf KO has no affect by itself so what is going on in this patient? Response: We have added to revised ms that there is no information available of any other underlying mutations contributing to the patient's hearing loss. Parents were heterozygous for MANF variants (Montaser et al., 2021). No information is available of their hearing status (results lines 298-301). We have extended this discussion in the revised ms (discussion lines 447-451). As we say, there is a future need to screen for MANF variants in the hearing loss databases, without the possible diabetes bias, which is the case with the few MANF patients thus far found.
8. The IHC controls in figure 3 should be shown since the expression patterns seem to be different to the OHCs. This should also be explained. Response: These data are shown in Fig. S3 in revised ms.
9. In figure 5N why is only one mouse used? Why not use both mice for this? The numbers of mice are very low anyway at n=2 for the analysis of TEM ribbon morphology so at least use all you have. Were the ribbons in CD-1 mice investigated at all? It would be interesting to see if the numbers are comparably low, although I understand there is no control for these. Response: We agree with the referee about the need for higher sample size. In the revision process, we added 2 cochleas of both genotype to the analysis (now n = 3 in both cases). We could not confirm the original finding of increased proportion of double ribbons in IHCs of mutant cochleas. The variability was too high for statistical power with three specimens. Therefore, we have omitted the double ribbon data from the revised ms. We would like to point out that the amount of thin TEM sections analyzed per cochlea is measured in the hundreds in our work.
We are currently investigating ribbon synapses of CD-1 mice in another project, by counting CtBP2-positive ribbons. We have not done TEM analysis with CD-1 mice. To briefly summarize, CD-1 wildtype mice suffer early-onset, severe cochlear synaptopathy.

Reviewer #3 (Comments to the Authors (Required)):
…one critical timing problem limits the impact of their conclusion, Specifically, they show that Manf cKO mice have significant hearing loss, particularly at high frequencies, as early as P15 (shortly after the onset of hearing). In fact, the high-frequency thresholds are largely the same at P15 and P56. However, they find evidence of subtle stereociliary splaying in both high-and low-frequency outer hair cells at P22 (though they do not show the images for the low-frequency OHCs), and only evidence of sterociliary fusion at P56. Therefore, the time course and frequency specificity of their stereociliary phenotype (splaying and/or fusion) does not match with the timing of the hearing phenotype. This calls into question any conclusion they may draw into the specific mechanistic role through which Manf deficiency causes hearing loss through structural changes in the stereocilia, which is a central point of the paper. Response: This is an astute observation and the question is important as hearing function was impaired in the mentioned young mice and, correspondingly, in the human patient. We have approached the question in revised manuscript with both new experiments and discussion. This referee addressed in essence similar questions as referee#2. Please see above the response to Editor#2 comment and the response to referee#2.
In addition to the information delivered in those responses: (1) We have added to revised ms images of the low-frequency OHC stereociliary splaying at the juvenile stage and at adulthood (revised ms Figs S1, 4B and F). We have gathered all data on hearing measurements to the same figure (revised ms Fig 1), allowing the reader to follow hearing deterioration in time. If you are planning a press release on your work, please inform us immediately to allow informing our production team and scheduling a release date.
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