Aldh inhibitor restores auditory function in a mouse model of human deafness

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Research Article

Guang-Jie Zhu ,

Sihao Gong ,

Deng-Bin Ma ,

Tao Tao ,

Wei-Qi He ,

Linqing Zhang,

Fang Wang,

Xiao-Yun Qian,

Han Zhou,

Chi Fan,

Pei Wang,

Xin Chen,

Wei Zhao,

Jie Sun,

Huaqun Chen,

Ye Wang,

Xiang Gao,

Jian Zuo,

Min-Sheng Zhu ,

Xia Gao ,

 [ … ],

Guoqiang Wan

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Guang-Jie Zhu, 

Sihao Gong, 

Deng-Bin Ma, 

Tao Tao, 

Wei-Qi He, 

Linqing Zhang, 

Fang Wang, 

Xiao-Yun Qian, 

Han Zhou, 

Chi Fan


Published: September 24, 2020


?This is an uncorrected proof.

AbstractGenetic hearing loss is a common health problem with no effective therapy currently available. DFNA15, caused by mutations of the transcription factor POU4F3, is one of the most common forms of autosomal dominant non-syndromic deafness. In this study, we established a novel mouse model of the human DFNA15 deafness, with a Pou4f3 gene mutation (Pou4f3Δ) identical to that found in a familial case of DFNA15. The Pou4f3(Δ/+) mice suffered progressive deafness in a similar manner to the DFNA15 patients. Hair cells in the Pou4f3(Δ/+) cochlea displayed significant stereociliary and mitochondrial pathologies, with apparent loss of outer hair cells. Progression of hearing and outer hair cell loss of the Pou4f3(Δ/+) mice was significantly modified by other genetic and environmental factors. Using Pou4f3(-/+) heterozygous knockout mice, we also showed that DFNA15 is likely caused by haploinsufficiency of the Pou4f3 gene. Importantly, inhibition of retinoic acid signaling by the aldehyde dehydrogenase (Aldh) and retinoic acid receptor inhibitors promoted Pou4f3 expression in the cochlear tissue and suppressed the progression of hearing loss in the mutant mice. These data demonstrate Pou4f3 haploinsufficiency as the main underlying cause of human DFNA15 deafness and highlight the therapeutic potential of Aldh inhibitors for treatment of progressive hearing loss.
Author summary
More than 50% of deafness cases are due to genetic defects with no treatment available. DFNA15, caused by mutations of the transcription factor POU4F3, is one of the most common types of autosomal dominant non-syndromic deafness. Here, we established a novel mouse model with the exact Pou4f3 mutation identified in human patients. The mutant mouse display similar auditory pathophysiology as human patients and exhibit multiple hair cell abnormalities. The onset and severity of hearing loss in the mouse model is highly modifiable to environmental factors, such as aging, noise exposure or genetic backgrounds. Using a new knockout mouse model, we found Pou4f3 haploinsufficiency as the underlying mechanism of human DFNA15. Importantly, we identified Aldh inhibitor as a potent small molecule for upregulation of Pou4f3 and treatment of hearing loss in the mutant mouse. The identification of Aldh inhibitor for treatment of DFNA15 deafness represents a major advance in the unmet medical need for this common form of progressive hearing loss.

Citation: Zhu G-J, Gong S, Ma D-B, Tao T, He W-Q, Zhang L, et al. (2020) Aldh inhibitor restores auditory function in a mouse model of human deafness. PLoS Genet 16(9):

https://doi.org/10.1371/journal.pgen.1009040Editor: Lisa Cunningham, National Institutes of Health, UNITED STATESReceived: April 14, 2020; Accepted: August 10, 2020; Published: September 24, 2020Copyright: © 2020 Zhu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Data Availability: The RNA-seq data are available at GEO database with accession number GSE139145.Funding: The research is supported by the National Natural Science Funding of China (Project IDs: 81371090, 31330034, 31671548 to MSZ and Project ID: 31771153 to GW; www.nsfc.gov.cn). The study is also funded by the National Institutes of Health (Project ID: R01DC006471 to JZ; www.nih.gov). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing interests: The authors have declared that no competing interests exist.

IntroductionHearing loss is one of the most common sensory defects resulting from both genetic and environmental insults that affect more than 250 million people worldwide [1]. Despite the prevalence of hearing loss, no Food and Drug Administration-approved therapeutic treatment is currently available. Among the genetic hearing disorders, 20% of the cases are the autosomal dominant inheritance of nonsyndromic hearing loss [2]. POU4F3 mutations are associated with DFNA15, one of the common autosomal dominant forms of progressive hearing loss [3–5]. Studies using the Pou4f3-null mice indicate that Pou4f3 plays an essential role in differentiation and survival of inner ear hair cells [6–8]. However, how Pou4f3 mutations in human patients cause the dominant form of progressive hearing loss remains unknown.
To shed light into the pathology and molecular basis of DFNA15 progressive hearing loss, we generated a novel mouse model harboring the exact human POU4F3 mutation identified in the first identified familial case of DFNA15 [3]. In this study, using a combination of Pou4f3 mutant and knockout animal models, we examined the pathophysiology and hair cell histopathology of DFNA15 deafness, revealed Pou4f3 haploinsufficiency as the main underlying cause of the deafness, and identified retinoic acid signaling inhibitors for Pou4f3 upregulation and treatment of DFNA15 deafness.

The DFNA15 mouse model displays progressive hearing loss
We established a knock-in mouse line with an 8bp deletion and a C-T reversion of the mouse Pou4f3 gene (Pou4f3Δ; S1A and S1B Fig), which was identical to the POU4F3 mutation found in an Israeli Jewish DFNA15 patient family [3]. This mutation of Pou4f3 in the target ES cells was confirmed by Southern blot analysis and sequencing (S1C Fig). Genotyping analysis of Pou4f3(Δ/+) mice with tail genomic DNA showed a specific PCR product (542 bp), suggesting successful germ line transmission of the mutation (S1D Fig). Previous in vitro studies demonstrate that the 8bp deletion of Pou4f3 gene results in a truncated Pou4f3 protein with impaired nuclear localization [9, 10]. Western blot of P3 cochleae from wildtype (+/+), heterozygous (Δ/+) and homozygous (Δ/Δ) cochlea also showed a smaller band of the truncated Pou4f3 protein in the cochlea of Pou4f3(Δ/+) and Pou4f3(Δ/Δ) mice (S1E Fig). When we expressed wildtype Pou4f3 (Pou4f3-wt) and the mutant Pou4f3 (Pou4f3-8del) in HeLa cells, mutant Pou4f3 also displayed impaired nuclear localization (S1F Fig). Similarly, Pou4f3 immunostaining of P10 wildtype and mutant cochleae showed cytoplasmic localization of the mutant Pou4f3 and weaker nuclear Pou4f3 signal in outer hair cells of the Pou4f3(Δ/+) cochleae (S1G Fig).
Human DFNA15 deafness presents as post-lingual progressive hearing loss [3, 11]. We then measured auditory functions of 2–7 months old Pou4f3(Δ/+) mice using distortion product otoacoustic emissions (DPOAEs) and auditory brainstem responses (ABRs). Both DPOAE (Fig 1A) and ABR thresholds (Fig 1B) of Pou4f3(Δ/+) mice were comparable to the wildtype mice at 2 months age. However, ABR peak 1 (P1) amplitudes of the Pou4f3(Δ/+) mice were significantly reduced at 32 kHz (Fig 1C and S2 Fig), indicating that these mutant mice start to display mild auditory phenotype at 2 months. As mice aged further to 3 months (Fig 1D–1F and 1J) and 7 months (Fig 1G–1I), DPOAE thresholds, ABR thresholds and ABR P1 amplitudes of the Pou4f3(Δ/+) mice altered significantly and progressively from high to low cochlear frequencies (Fig 1 and S2 Fig). This observation suggests that Pou4f3(Δ/+) mice displayed an adult-onset progressive hearing loss that starts at high frequencies, consistent with the progression of human DFNA15 [11]. In contrast to the wildtype or heterozygous Pou4f3(Δ/+) mice, the homozygous Pou4f3(Δ/Δ) were profoundly deaf (S3A Fig) and lost all hair cells (S3B Fig) within the first month of age. This remarkable phenotype resembles that observed in Pou4f3 knockout mice [12, 13], suggesting that hair cell survival is critically dependent on Pou4f3 function. Our result also indicates that Pou4f3 plays a dosage-dependent role in auditory function.
Fig 1. Pou4f3(Δ/+) mice display progressive hearing loss.(A-C) 2 months (n=9–10), (D-F) 3 months (n=14–16) and (G-I) 7 months (n=8 each) old wildtype Pou4f3(+/+) and mutant Pou4f3(Δ/+) mice were tested with DPOAE and ABR. (A, D, G) DPOAE thresholds; (B, E, H) ABR thresholds; (C, F, I) ABR peak 1 (P1) amplitudes. * P Read More

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