Repressor element 1-silencing transcription factor deficiency yields profound hearing loss through Kv7.4 channel upsurge in auditory neurons and hair cells
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Evaluation Summary:
Genetic forms of deafness are a major health challenge. This study deciphers the cochlear roles of Repressor element 1-silencing transcription factor (REST), a gene involved in the DFNA27 dominant form of deafness, using the mouse as a model system. This study provides evidence for a pathophysiological mechanism of deafness and shows how genes involved in different forms of deafness may interact together. The manuscript will be interesting to readers who work in the field of hearing research, REST regulation, or Kv7.4 regulation.
(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 #2 and Reviewer #3 agreed to share their names with the authors.)
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Abstract
Repressor element 1-silencing transcription factor (REST) is a transcriptional repressor that recognizes neuron-restrictive silencer elements in the mammalian genomes in a tissue- and cell-specific manner. The identity of REST target genes and molecular details of how REST regulates them are emerging. We performed conditional null deletion of Rest (cKO), mainly restricted to murine hair cells (HCs) and auditory neurons (aka spiral ganglion neurons [SGNs]). Null inactivation of full-length REST did not affect the development of normal HCs and SGNs but manifested as progressive hearing loss in adult mice. We found that the inactivation of REST resulted in an increased abundance of K v 7.4 channels at the transcript, protein, and functional levels. Specifically, we found that SGNs and HCs from Rest cKO mice displayed increased K v 7.4 expression and augmented K v 7 currents; SGN’s excitability was also significantly reduced. Administration of a compound with K v 7.4 channel activator activity, fasudil, recapitulated progressive hearing loss in mice. In contrast, inhibition of the K v 7 channels by XE991 rescued the auditory phenotype of Rest cKO mice. Previous studies identified some loss-of-function mutations within the K v 7.4-coding gene, Kcnq4 , as a causative factor for progressive hearing loss in mice and humans. Thus, the findings reveal that a critical homeostatic K v 7.4 channel level is required for proper auditory functions.
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Evaluation Summary:
Genetic forms of deafness are a major health challenge. This study deciphers the cochlear roles of Repressor element 1-silencing transcription factor (REST), a gene involved in the DFNA27 dominant form of deafness, using the mouse as a model system. This study provides evidence for a pathophysiological mechanism of deafness and shows how genes involved in different forms of deafness may interact together. The manuscript will be interesting to readers who work in the field of hearing research, REST regulation, or Kv7.4 regulation.
(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 #2 and Reviewer #3 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
In this study, Haiwei Zhang and colleagues test the effects of cell type-specific deletion of the transcriptional repressor REST on Kv7.4 ion channel activity in the cochlea and hearing. To achieve cell type-specific deletion of Rest (Rest cKO), they intercrossed two previously generated mouse lines: one that is homozygous for a floxed allele of Rest, and another that is hemizygous for a Cre-encoding transgene (i.e., Tg(Atoh1-Cre)). This transgene is expressed mainly (but not exclusively) in hair cells (HCs) and spiral ganglion neurons (SGNs) in the inner ear. The generated Rest cKO mice were used for a variety of tests, i.e., hearing tests, histological analysis of the cochlea, qRT-PCR tests, and electrophysiological tests. Based on these tests and analyses, the authors conclude that REST-dependent …
Reviewer #1 (Public Review):
In this study, Haiwei Zhang and colleagues test the effects of cell type-specific deletion of the transcriptional repressor REST on Kv7.4 ion channel activity in the cochlea and hearing. To achieve cell type-specific deletion of Rest (Rest cKO), they intercrossed two previously generated mouse lines: one that is homozygous for a floxed allele of Rest, and another that is hemizygous for a Cre-encoding transgene (i.e., Tg(Atoh1-Cre)). This transgene is expressed mainly (but not exclusively) in hair cells (HCs) and spiral ganglion neurons (SGNs) in the inner ear. The generated Rest cKO mice were used for a variety of tests, i.e., hearing tests, histological analysis of the cochlea, qRT-PCR tests, and electrophysiological tests. Based on these tests and analyses, the authors conclude that REST-dependent 'moderation' of Kv7.4 expression is critical in HCs and SGNs for hearing. This conclusion is interesting because it is the ~inverse of what is currently known about REST and Kv7.4. Specifically, alternative splicing-dependent suppression of REST activity is known to be important in HCs for hearing, and loss-of-function mutations (but not gain-of-function mutations) in the Kv7.4-encoding gene have been shown to cause hearing loss. The weaknesses of the manuscript are related to the experimental design of the analysis of cochlear REST expression, the Tg(Atoh1-Cre) expression pattern, the Kv7.4-unrelated effects of fasudil (which was used to activate Kv7.4 in vivo), and the lack of genetic evidence for the proposed hearing loss-causing effect of abnormally high Kv7.4 expression.
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Reviewer #2 (Public Review):
Zhang and colleagues show that the repressor element 1 silencing transcription factor (REST) regulates the expression of Kv7 potassium channels in the inner ear. REST knockout mice have elevated hearing thresholds and altered mechanics as probed by ABR and DPOAE thresholds. The authors combine patch-clamp recordings with pharmacological manipulation of Kv7 channels, single-cell RT-PCR, and immunohistochemistry to convincingly attribute the functional changes in auditory thresholds to an upregulation of Kv7.4 channels and Kv7 mediated potassium currents in both hair cells and auditory neurons in REST knockout animals. The data are clearly presented.
By the third post-natal month, hair cells and SGN deteriorated in REST knockouts. The authors argue that this deterioration and a resulting progressive increase …
Reviewer #2 (Public Review):
Zhang and colleagues show that the repressor element 1 silencing transcription factor (REST) regulates the expression of Kv7 potassium channels in the inner ear. REST knockout mice have elevated hearing thresholds and altered mechanics as probed by ABR and DPOAE thresholds. The authors combine patch-clamp recordings with pharmacological manipulation of Kv7 channels, single-cell RT-PCR, and immunohistochemistry to convincingly attribute the functional changes in auditory thresholds to an upregulation of Kv7.4 channels and Kv7 mediated potassium currents in both hair cells and auditory neurons in REST knockout animals. The data are clearly presented.
By the third post-natal month, hair cells and SGN deteriorated in REST knockouts. The authors argue that this deterioration and a resulting progressive increase in thresholds is due to Kv7 upregulation. Since REST knockouts may influence more than Kv7 channels, I found this argument to be less convincing.
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Reviewer #3 (Public Review):
Genetic forms of deafness are a major health challenge. The causal genes for at least 140 isolated (non-syndromic) and 400 syndromic forms have already been uncovered. The study by Haiwei Zhang et al. deciphers the cochlear roles of Repressor element 1-silencing transcription factor (REST), a gene involved in the DFNA27 dominant form of deafness. The authors showed that the conditional inactivation of the Rest encoding gene in the cochlea and auditory nerve led to progressive deafness in mice. Rest, as a repressor element, has been shown to regulate the expression level of many neuronal genes. The authors could show that the inactivation of Rest upregulates the expression of Kv7.4, a channel protein encoded by Kcnq4 and involved in another genetic form of deafness. As a consequence, the function of sensory …
Reviewer #3 (Public Review):
Genetic forms of deafness are a major health challenge. The causal genes for at least 140 isolated (non-syndromic) and 400 syndromic forms have already been uncovered. The study by Haiwei Zhang et al. deciphers the cochlear roles of Repressor element 1-silencing transcription factor (REST), a gene involved in the DFNA27 dominant form of deafness. The authors showed that the conditional inactivation of the Rest encoding gene in the cochlea and auditory nerve led to progressive deafness in mice. Rest, as a repressor element, has been shown to regulate the expression level of many neuronal genes. The authors could show that the inactivation of Rest upregulates the expression of Kv7.4, a channel protein encoded by Kcnq4 and involved in another genetic form of deafness. As a consequence, the function of sensory cells and the excitability of primary sensory neurons of the auditory nerve are impaired. The authors could further link Kv7.4 upregulation and the associated functional deficits in Rest conditional knock-out mice by showing that, on one hand, application of the Kv7.4 channel blocker XE991, rescued the auditory phenotype in Rest conditional knock-out mice, and on the other hand, application of the Kv7.4 activator fasudil, replicated in wild-type mice the hearing impairment observed in Rest conditional knock-out mice.
The authors have convincingly demonstrated the relationship between Rest cochlear inactivation and the upregulation of Kv7.4 leading to progressive hearing loss using a genetic model and also pharmacological experiments. These findings are important in the field of auditory research for several reasons. This study elucidates another pathophysiological mechanism of deafness. In addition, it shows how genes involved in different forms of deafness may interact together and how their associated deafness may be in fact linked in some patients. The pharmacological study may give new hints for pharmacological regulation of Kv7.4 activity in patients. Finally, a major challenge in the upcoming years will be the development of gene therapies implying a fine dissection of gene regulatory networks to rescue defective genes in specific cell types. The study of Rest in the cochlea provides new insight into the genes regulating the expression of other key genes for cochlear function. I have no major concerns. The study has generally been well designed and conducted.
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