Repair of noise-induced damage to stereocilia F-actin cores is facilitated by XIRP2 and its novel mechanosensor domain
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Evaluation Summary:
This study investigates a process by which hair cell stereocilia, the sensory structures that respond to sound in the hearing organ and to head motion or tilt in the vestibular organ, can recover from damage-induced gaps in their actin core, possibly allowing for the rescue of transient hearing loss after exposure to noise. This manuscript will be of strong interest to the inner ear field as well as readers with broader interest in actin cytoskeleton dynamics. Although meticulous controls, a combination of molecular, histological and functional studies and an innovative mouse model generally support the major conclusions of this study, additional controls are needed to confirm the mechanistic claims made in the manuscript.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Prolonged exposure to loud noise has been shown to affect inner ear sensory hair cells in a variety of deleterious manners, including damaging the stereocilia core. The damaged sites can be visualized as ‘gaps’ in phalloidin staining of F-actin, and the enrichment of monomeric actin at these sites, along with an actin nucleator and crosslinker, suggests that localized remodeling occurs to repair the broken filaments. Herein, we show that gaps in mouse auditory hair cells are largely repaired within 1 week of traumatic noise exposure through the incorporation of newly synthesized actin. We provide evidence that Xin actin binding repeat containing 2 (XIRP2) is required for the repair process and facilitates the enrichment of monomeric γ-actin at gaps. Recruitment of XIRP2 to stereocilia gaps and stress fiber strain sites in fibroblasts is force-dependent, mediated by a novel mechanosensor domain located in the C-terminus of XIRP2. Our study describes a novel process by which hair cells can recover from sublethal hair bundle damage and which may contribute to recovery from temporary hearing threshold shifts and the prevention of age-related hearing loss.
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Evaluation Summary:
This study investigates a process by which hair cell stereocilia, the sensory structures that respond to sound in the hearing organ and to head motion or tilt in the vestibular organ, can recover from damage-induced gaps in their actin core, possibly allowing for the rescue of transient hearing loss after exposure to noise. This manuscript will be of strong interest to the inner ear field as well as readers with broader interest in actin cytoskeleton dynamics. Although meticulous controls, a combination of molecular, histological and functional studies and an innovative mouse model generally support the major conclusions of this study, additional controls are needed to confirm the mechanistic claims made in the manuscript.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers …
Evaluation Summary:
This study investigates a process by which hair cell stereocilia, the sensory structures that respond to sound in the hearing organ and to head motion or tilt in the vestibular organ, can recover from damage-induced gaps in their actin core, possibly allowing for the rescue of transient hearing loss after exposure to noise. This manuscript will be of strong interest to the inner ear field as well as readers with broader interest in actin cytoskeleton dynamics. Although meticulous controls, a combination of molecular, histological and functional studies and an innovative mouse model generally support the major conclusions of this study, additional controls are needed to confirm the mechanistic claims made in the manuscript.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In their manuscript "Repair of Noise-Induced Damage to Stereocilia F-actin Cores is Facilitated by XIRP2," Wagner et al. make a significant contribution to understanding how noise-induced damage to stereocilia is repaired. Through immunostaining of XIRP2 in isolated mouse inner hair cells (IHCs) exposed to noise-induced damage, as well as IHCs isolated from human patients, the authors clearly demonstrate the recruitment of XIRP2 to sites of damage within the stereocilia F-actin core (referred to as "gaps"). These gaps are ultimately repaired and filled in with actin monomers. They furthermore show through immunostaining that Xirp2 knockout IHCs lack the recruitment of γ-actin to gaps. Notably, the authors identify the enrichment of the short isoform of XIRP2 at gaps, but not the long isoform through …
Reviewer #1 (Public Review):
In their manuscript "Repair of Noise-Induced Damage to Stereocilia F-actin Cores is Facilitated by XIRP2," Wagner et al. make a significant contribution to understanding how noise-induced damage to stereocilia is repaired. Through immunostaining of XIRP2 in isolated mouse inner hair cells (IHCs) exposed to noise-induced damage, as well as IHCs isolated from human patients, the authors clearly demonstrate the recruitment of XIRP2 to sites of damage within the stereocilia F-actin core (referred to as "gaps"). These gaps are ultimately repaired and filled in with actin monomers. They furthermore show through immunostaining that Xirp2 knockout IHCs lack the recruitment of γ-actin to gaps. Notably, the authors identify the enrichment of the short isoform of XIRP2 at gaps, but not the long isoform through antibodies specific to each isoform. The authors identified a predicted LIM domain in the C-terminal sequence of the short isoform of XIRP2. Truncation of the short isoform of XIRP2 (Xirp2-ΔCterm) resulted in a loss of γ-actin recruitment to gaps, phenocopying Xirp2 knockout mice. Finally, the authors clearly show that Xirp2 knockout mice are more susceptible to various types of hearing loss through hearing tests, including noise-induced hearing loss. In sum, their experiments support a mechanism by which the short isoform of XIRP2 is localized to gaps in stereocilia and facilitates repair by recruiting γ-actin to fill them. Repair of these gaps significantly contributes to stereocilia maintenance and prevention of permanent hearing loss in mice. Overall, the experimental work presented is convincing, and strongly supports a requirement for the XIRP2 short isoform in repairing stereocilia gaps. However, a substantive concern is that while the authors extensively suggest that XIRP2 likely employs a mechanism similar to other LIM domain-containing proteins for repairing mechanical damage to actin bundles, the evidence provided to support this claim is modest.
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Reviewer #2 (Public Review):
The 'hair' cells of the inner ear and their sensory apparatuses, called stereocilia, are non-renewable but have a limited capacity for self-repair in mammals. Following exposure to noise, a temporary reduction in hearing sensitivity can be followed by recovery. Repair of inter-stereocilia 'tip links' was proposed to contribute to this phenomenon, for example. Noise-induced actin gaps in the shaft of the stereocilia have been noted as well, but have been little studied to date. Here, Wagner et al. pick up on a story left off over a decade ago, in which Belyantseva et al. (2009) noted that damage-induced gaps in stereocilia may undergo actin remodeling, suggesting a new mode of hair cell repair. In this new manuscript, Wagner et al. sought to provide bona fide evidence of actin gap repair and to delve into the …
Reviewer #2 (Public Review):
The 'hair' cells of the inner ear and their sensory apparatuses, called stereocilia, are non-renewable but have a limited capacity for self-repair in mammals. Following exposure to noise, a temporary reduction in hearing sensitivity can be followed by recovery. Repair of inter-stereocilia 'tip links' was proposed to contribute to this phenomenon, for example. Noise-induced actin gaps in the shaft of the stereocilia have been noted as well, but have been little studied to date. Here, Wagner et al. pick up on a story left off over a decade ago, in which Belyantseva et al. (2009) noted that damage-induced gaps in stereocilia may undergo actin remodeling, suggesting a new mode of hair cell repair. In this new manuscript, Wagner et al. sought to provide bona fide evidence of actin gap repair and to delve into the mechanism by which these gaps can be resolved.
Wagner et al. show that noise-induced stereocilia actin gaps are repaired within 1 week following noise exposure, in itself an important new contribution addressing a long-standing question in the field. They also leverage two mutant mouse strains as genetic susceptibility models that show gaps without noise-exposure, broadening the importance of gaps and repair. They show that the actin binding protein XIRP2 is enriched in actin gaps in mouse auditory and vestibular hair cells, as well as human vestibular hair cells, and is able to bind actin monomers. Remarkably, XIRP2 knock-out mutants have a reduced ability to repair gaps, to accumulate monomeric actin at gap sites, or to recover their hearing post-noise damage as compared to control mice. Having identified XIRP2 short isoform as the protein form enriched at gaps, the authors engineer a clever XIRP2 mutant mouse line that specifically targets the C-terminus and LIM (putative actin-binding) domain of the short isoform, presumably leaving other XIRP2 functions intact. This model has the potential to specifically ablate XIRP2's gap filling behavior, and thus to assess directly how gap repair impacts hearing upon noise challenges.
The authors provide painstaking controls to ensure that the observed reduction in actin gaps post-noise over time is due to gap repair, not hair cell death or stereocilia loss. In general, the authors achieved their goals of verifying the hypothesis that stereocilia actin gaps can be repaired after damage, and provided strong support that XIRP2 is required for actin gap repair.
However several areas can be improved to further strengthen the manuscript. Among others, the gamma-actin immunolabeling raises some questions, as signal is absent at stereocilia shafts and the antibody used does not seem to be characterized. The XIRP2 DelCter mutant is used to show lack of XIRP2 and actin enrichment at gaps, but not to verify the prediction of an excess number of gaps and defective ABR threshold recovery in this model. The manuscript would also greatly benefit from modifications and additions in data reporting.
Overall, this study provides a substantial and long-awaited contribution to our limited understanding of how mammalian hair cells maintain their precise cellular architecture over a lifetime of wear.
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Reviewer #3 (Public Review):
Wagner et al. address a form of damage to the bundle of F-actin filaments that comprises the core of stereocilia, which are mechanosensitive protrusions on the surface of auditory and vestibular sensory cells. The authors detect breaks in the F-actin core, primarily occurring following noise exposure in auditory stereocilia and for unknown reasons in vestibular stereocilia. Following noise damage, the number of gaps in auditory stereocilia increases within an hour and then decreases back to baseline over two weeks. Newly synthesized GFP-actin integrates into the stereocilia core, which is otherwise stably maintained with little to no actin turnover.
Similar breaks in stereocilia were previously known to contain monomeric actin, espin, and cofilin. The authors have added the crosslinker XIRP2 to the list, …
Reviewer #3 (Public Review):
Wagner et al. address a form of damage to the bundle of F-actin filaments that comprises the core of stereocilia, which are mechanosensitive protrusions on the surface of auditory and vestibular sensory cells. The authors detect breaks in the F-actin core, primarily occurring following noise exposure in auditory stereocilia and for unknown reasons in vestibular stereocilia. Following noise damage, the number of gaps in auditory stereocilia increases within an hour and then decreases back to baseline over two weeks. Newly synthesized GFP-actin integrates into the stereocilia core, which is otherwise stably maintained with little to no actin turnover.
Similar breaks in stereocilia were previously known to contain monomeric actin, espin, and cofilin. The authors have added the crosslinker XIRP2 to the list, showing compelling immunofluorescent localization data placing XIRP2 either in or at either edge of the break. XIRP2 knockout mice have more stereocilia breaks in both auditory and vestibular stereocilia. In auditory stereocilia the number of breaks steadily increases after noise damage over the normal recovery period, suggesting repair requires XIRP2. The mechanism may involve recruiting XIRP2 associated with monomeric gamma-actin, which is reduced in breaks in XIRP2 knockout vestibular stereocilia. XIRP2 is proposed to bind damaged F-actin in breaks via its C-terminal LIM domains. Correspondingly, XIRP2 lacking these LIM domains does not localize to breaks in vestibular stereocilia and the breaks also have lower levels of gamma-actin.
This paper provides significant new insights into the generation and resolution of breaks in the F-actin core, establishing the general time course of repair following noise damage and showing that repair proceeds by an infill mechanism rather than wholesale replacement of stereocilia actin. The molecular process by which these patches arise is still unclear, but the data implicate XIRP2.
The authors present a very interesting hypothesis regarding how XIRP2 functions, which is that XIRP2 is recruited to strained actin filaments in damaged regions of the stereocilia core via its C-terminal LIM domains, where it recruits monomeric actin used to repair broken filaments. Showing that XIRP2 LIM domains bound strained actin filaments and/or that strained filaments existed in the stereocilia breaks would make this hypothesis more compelling, as would demonstrating that XIRP2 recruited G-actin to breaks in noise-damaged auditory stereocilia as well as vestibular stereocilia.
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