The pathogenesis of common Gjb2 mutations associated with human hereditary deafness in mice

doi:10.1007/s00018-023-04794-9

. 2023 May 13;80(6):148.

doi: 10.1007/s00018-023-04794-9.

The pathogenesis of common Gjb2 mutations associated with human hereditary deafness in mice

Qing Li^#¹, Chong Cui^#^{2

3

4}, Rongyu Liao^#⁵, Xidi Yin^#⁵, Daqi Wang^#^{2

3

4}, Yanbo Cheng⁶, Bowei Huang^{2

3

4}, Liqin Wang^{2

4}, Meng Yan⁷, Jinan Zhou^{2

3

4}, Jingjing Zhao^{2

4}, Wei Tang⁵, Yingyi Wang⁶, Xiaohan Wang⁸, Jun Lv^{2

3

4}, Jinsong Li^{5

6

7}, Huawei Li^{9

10

11}, Yilai Shu^{12

13

14}

Affiliations

¹ State Key Laboratory of Cell Biology, Shanghai Key Laboratory of Molecular Andrology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China. liqing2015@sibcb.ac.cn.
² ENT Institute and Department of Otorhinolaryngology, Eye and ENT Hospital, State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Fudan University, Shanghai, China.
³ Institutes of Biomedical Sciences, Fudan University, Shanghai, China.
⁴ NHC Key Laboratory of Hearing Medicine, Fudan University, Shanghai, China.
⁵ State Key Laboratory of Cell Biology, Shanghai Key Laboratory of Molecular Andrology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China.
⁶ School of Life Science and Technology, Shanghai Tech University, Shanghai, China.
⁷ School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, China.
⁸ Renerval Biotherapeutics, Jiangsu, China.
⁹ ENT Institute and Department of Otorhinolaryngology, Eye and ENT Hospital, State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Fudan University, Shanghai, China. hwli@shmu.edu.cn.
¹⁰ Institutes of Biomedical Sciences, Fudan University, Shanghai, China. hwli@shmu.edu.cn.
¹¹ NHC Key Laboratory of Hearing Medicine, Fudan University, Shanghai, China. hwli@shmu.edu.cn.
¹² ENT Institute and Department of Otorhinolaryngology, Eye and ENT Hospital, State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Fudan University, Shanghai, China. yilai_shu@fudan.edu.cn.
¹³ Institutes of Biomedical Sciences, Fudan University, Shanghai, China. yilai_shu@fudan.edu.cn.
¹⁴ NHC Key Laboratory of Hearing Medicine, Fudan University, Shanghai, China. yilai_shu@fudan.edu.cn.

^# Contributed equally.

PMID: 37178259
PMCID: PMC10182940
DOI: 10.1007/s00018-023-04794-9

The pathogenesis of common Gjb2 mutations associated with human hereditary deafness in mice

Qing Li et al. Cell Mol Life Sci. 2023.

. 2023 May 13;80(6):148.

doi: 10.1007/s00018-023-04794-9.

Authors

Affiliations

¹ State Key Laboratory of Cell Biology, Shanghai Key Laboratory of Molecular Andrology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China. liqing2015@sibcb.ac.cn.
² ENT Institute and Department of Otorhinolaryngology, Eye and ENT Hospital, State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Fudan University, Shanghai, China.
³ Institutes of Biomedical Sciences, Fudan University, Shanghai, China.
⁴ NHC Key Laboratory of Hearing Medicine, Fudan University, Shanghai, China.
⁵ State Key Laboratory of Cell Biology, Shanghai Key Laboratory of Molecular Andrology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China.
⁶ School of Life Science and Technology, Shanghai Tech University, Shanghai, China.
⁷ School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Hangzhou, China.
⁸ Renerval Biotherapeutics, Jiangsu, China.
⁹ ENT Institute and Department of Otorhinolaryngology, Eye and ENT Hospital, State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Fudan University, Shanghai, China. hwli@shmu.edu.cn.
¹⁰ Institutes of Biomedical Sciences, Fudan University, Shanghai, China. hwli@shmu.edu.cn.
¹¹ NHC Key Laboratory of Hearing Medicine, Fudan University, Shanghai, China. hwli@shmu.edu.cn.
¹² ENT Institute and Department of Otorhinolaryngology, Eye and ENT Hospital, State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Fudan University, Shanghai, China. yilai_shu@fudan.edu.cn.
¹³ Institutes of Biomedical Sciences, Fudan University, Shanghai, China. yilai_shu@fudan.edu.cn.
¹⁴ NHC Key Laboratory of Hearing Medicine, Fudan University, Shanghai, China. yilai_shu@fudan.edu.cn.

^# Contributed equally.

PMID: 37178259
PMCID: PMC10182940
DOI: 10.1007/s00018-023-04794-9

Abstract

Mutations in GJB2 (Gap junction protein beta 2) are the most common genetic cause of non-syndromic hereditary deafness in humans, especially the 35delG and 235delC mutations. Owing to the homozygous lethality of Gjb2 mutations in mice, there are currently no perfect mouse models carrying Gjb2 mutations derived from patients for mimicking human hereditary deafness and for unveiling the pathogenesis of the disease. Here, we successfully constructed heterozygous Gjb2^+/35delG and Gjb2^+/235delC mutant mice through advanced androgenic haploid embryonic stem cell (AG-haESC)-mediated semi-cloning technology, and these mice showed normal hearing at postnatal day (P) 28. A homozygous mutant mouse model, Gjb2^{35delG/35delG}, was then generated using enhanced tetraploid embryo complementation, demonstrating that GJB2 plays an indispensable role in mouse placenta development. These mice exhibited profound hearing loss similar to human patients at P14, i.e., soon after the onset of hearing. Mechanistic analyses showed that Gjb2 35delG disrupts the function and formation of intercellular gap junction channels of the cochlea rather than affecting the survival and function of hair cells. Collectively, our study provides ideal mouse models for understanding the pathogenic mechanism of DFNB1A-related hereditary deafness and opens up a new avenue for investigating the treatment of this disease.

Keywords: 235delC; 35delG; CX26; CX30; Gap junction channels; Hair cell development; Hearing loss.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

**Fig. 1**
The generation of *Gjb2* 35delG and 235delC heterozygous mice. A and B Schematic diagram of the construction of 35delG (A) and 235delC (B) knock-in in *Gjb2*. The blue bases represent the sgRNA sequence. The PAM sequence and the deleted base are shown in red and orange, respectively. The donor sequence ligates to the pMD19T vector. C Experimental procedures for producing heterozygous mouse models carrying 35delG or 235delC of *Gjb2* through semi-cloning technology. D Images of *Gjb2*^+/35delG and *Gjb2*^+/235delC semi-cloned (SC) mice at six weeks. E Representative Sanger sequencing results of *Gjb2*^+/35delG and *Gjb2*^+/235delC SC mice. F The averaged ABR thresholds of the 4-week-old wild-type, *Gjb2*^+/35delG, and *Gjb2*^+/235delC mice at 4, 8, 16, 24, and 32 kHz. n represents the number of tested mice. These heterozygous mice are the offspring of multiple generations of backcrossed with C57BL/6J mice. Wild-type and heterozygous mice from the same litter. G and H The ABR wave I amplitudes (G) and latencies (H) of the 4-week-old wild-type, *Gjb2*^+/35delG, and *Gjb2*^+/235delC mice at 4, 8, 16, 24, and 32 kHz. Data are shown as the mean ± s.e.m of the indicated biological replicates. n.s, no significance, **P < 0.01 by Student’s unpaired two-sided t-test

**Fig. 2**
The generation of *Gjb2* 35delG homozygous mice with profound deafness. A Schematic diagram of the generation of *Gjb2* 35delG homozygous mice via tetraploid embryo complementation (created in BioRender.com). B Representative Sanger sequencing results of wild-type and *Gjb2*^{35delG/35delG} males. C ABR waveforms were recorded at 8 kHz in the wild-type and *Gjb2*^{35delG/35delG} mice at P35. The blue and red traces indicate the thresholds of wild-type and *Gjb2*^{35delG/35delG} males, respectively. The scale bar for all traces is shown. D The average ABR thresholds of the P35 wild-type and *Gjb2*^{35delG/35delG} mice at 4, 8, 16, 24, and 32 kHz. Upward arrows indicate that there were no ABR responses at 90 dB SPL for the corresponding frequencies, and the hearing threshold was recorded as 90 dB SPL. n represents the number of tested mice. E Representative confocal microscopy images of Myo7a and Sox2 immunofluorescence in the apical, middle, and basal turns of the cochleae from wild-type and *Gjb2*^{35delG/35delG} mice at P35. White arrows indicate the loss of hair cells. Scale bar, 20 μm. F Quantification of surviving IHCs, OHCs, supporting cells of Deiters’ cells and outer/inner pillar cells (DCs and PCs) in the wild-type and *Gjb2*^{35delG/35delG} males at P35, respectively. Individual values are shown. Data are shown as the mean ± s.e.m of the indicated biological replicates. n.s, no significance, **P < 0.01, ***P < 0.001 by Student’s unpaired two-sided t-test

**Fig. 3**
The influence of GJB2 in the cochlea at P14. A ABR waveforms were recorded at 8 kHz in the wild-type and *Gjb2*^{35delG/35delG} males at P14. The blue and red traces indicate the thresholds of wild-type and *Gjb2*^{35delG/35delG} mice, respectively. The scale bar for all traces is shown. B The average ABR thresholds of the P14 wild-type and *Gjb2*^{35delG/35delG} males at 4, 8, 16, 24, and 32 kHz. Upward arrows indicate that there were no ABR responses at 90 dB SPL for the corresponding frequencies, and the hearing threshold was recorded as 90 dB SPL. n represents the number of tested mice. Data are shown as the mean ± s.e.m. *** P < 0.001 by Student’s unpaired two-sided t-test. C Spectra of DPOAE were recorded from the wild-type (blue trace) and *Gjb2*^{35delG/35delG} (red trace) males at P14. f₀ = 16 kHz, f2/f1 = 1.2, L₁/L₂ = 60/60 dB SPL. The peak of DPOAE (2f₁–f₂) in *Gjb2*^{35delG/35delG} mice was not available but f1 and f2 peaks remained the same as those in wild-type mice. D The average DPOAE thresholds were measured on the same animals performed with ABR recording at 4, 8, 16, 24, and 32 kHz. Upward arrows indicate that there were no DPOAE responses at the stimulus level of 80 dB SPL for the corresponding frequencies, and the DPOAE threshold was recorded as 80 dB SPL. n represents the number of tested mice. Data are shown as the mean ± s.e.m. **** P < 0.0001 by Student’s unpaired two-sided t-test. E Input/output function of the DPOAE (2f₁–f₂) amplitudes of wild-type and *Gjb2*^{35delG/35delG} males at 16 kHz frequency at P14. Data are shown as the mean ± s.e.m. The dotted line is the noise level. F Representative confocal microscopy images of Myo7a and Sox2 immunofluorescence in the apical, middle, and basal turns of the cochleae from wild-type and *Gjb2*^{35delG/35delG} males at P14. Scale bar, 20 μm. G Quantification of surviving inner hair cells (IHCs), outer hair cells (OHCs), supporting cells of Deiters’ cells and outer/inner pillar cells (DCs and PCs) in the wild-type and Gjb2^{35delG/35delG} males at P14, respectively. Data are shown as the mean ± s.e.m of the indicated biological replicates. n.s., no significance by Student’s unpaired two-sided t-test

**Fig. 4**
The mutation of GJB2 disrupts the formation of GJCs by reducing the GJB6 protein. A The panoramic view of representative confocal images of apical turns from wild-type and 35delG homozygous mice at P14, by staining GJB2, GJB6, and Sox2. The regions containing different supporting cells were delineated in the figure. Scale bar, 20 μm. B The diagram of the organ of Corti. C Representative confocal microscopy images of GJB2 and GJB6 immunofluorescence in the apical, middle, and basal turns of the cochleae from wild-type and *Gjb2*^{35delG/35delG} mice at P14. The inner sulcus areas were shown. Three independent mice were analyzed for each group. Scale bar, 20 μm. The bottom row shows high magnification views of GJB6. D Western blots showing GJB2 and GJB6 protein expression levels in the different tissues of wild-type and *Gjb2*^{35delG/35delG} mice, including cerebellum, liver, bladder, tail, and cochlea. Results obtained with GADPH are shown as controls for the amount of protein loading in each lane. Two biological repeats for each group. E and F Transcriptional analysis of *Gjb2* (E) and *Gjb6* (F) genes in different tissues of wild-type and *Gjb2*^{35delG/35delG} mice by qPCR, including cerebellum, liver, bladder, tail, and cochlea. Three biological repeats for each group. The expression values were normalized to that of *Gapdh*. Data are shown as the average mean ± s.e.m. *P < 0.05; n.s, no significant difference

**Fig. 5**
The disruption of GJCs in homozygous 35delG impairs the structure and function of cochlea. A Volcano plot showing the DEGs between *Gjb2*^{35delG/35delG} and wild-type cochleae in the apical, middle, and basal turns (adjusted P-value < 0.01 and fold changes > 2). B The KEGG pathway analysis of DEGs between wild-type and *Gjb2*^{35delG/35delG} cochleae in the middle and basal turns. C GO analysis of down-regulated genes between *Gjb2*^{35delG/35delG} and wild-type cochleae in the middle and basal turns. D Representative TEM images of the tunnel of Corti in the middle turn of the cochleae from wild-type and *Gjb2*^{35delG/35delG} mice at P14. The right images show the high magnification (1400 × magnification) views of the left black box (460 × magnification). Two independent mice were analyzed for each group. Scale bar: 10 μm. TC, tunnel of Corti; NS, Nuel's space; IHC, inner hair cell; OHC, outer hair cell; IPC, inner pillar cell; OPC, outer pillar cell; DC, Deiters’ cell. E Representative EP recordings from the cochleae of wild-type and *Gjb2*^{35delG/35delG} mice at P35. F The average EP magnitudes from the cochleae of wild-type and *Gjb2*^{35delG/35delG} mice at P14 and P35. Data are shown as the mean ± s.e.m of the indicated biological replicates. n.s, no significance, ***P < 0.001 by Student’s unpaired two-sided t-test

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References

1. Smith RJ, Bale JF, Jr, White KR. Sensorineural hearing loss in children. Lancet. 2005;365(9462):879–890. - PubMed
1. Morton CC, Nance WE. Newborn hearing screening–a silent revolution. N Engl J Med. 2006;354(20):2151–2164. - PubMed
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1. Chan DK, Chang KW. GJB2-associated hearing loss: systematic review of worldwide prevalence, genotype, and auditory phenotype. Laryngoscope. 2014;124(2):E34–53. - PubMed

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[1] Smith RJ, Bale JF, Jr, White KR. Sensorineural hearing loss in children. Lancet. 2005;365(9462):879–890. - PubMed

[2] Smith RJ, Bale JF, Jr, White KR. Sensorineural hearing loss in children. Lancet. 2005;365(9462):879–890. - PubMed

[3] Morton CC, Nance WE. Newborn hearing screening–a silent revolution. N Engl J Med. 2006;354(20):2151–2164. - PubMed

[4] Morton CC, Nance WE. Newborn hearing screening–a silent revolution. N Engl J Med. 2006;354(20):2151–2164. - PubMed

[5] Kokotas H, Petersen MB, Willems PJ. Mitochondrial deafness. Clin Genet. 2007;71(5):379–391. - PubMed

[6] Kokotas H, Petersen MB, Willems PJ. Mitochondrial deafness. Clin Genet. 2007;71(5):379–391. - PubMed

[7] Korver AM, Smith RJ, Van Camp G, Schleiss MR, Bitner-Glindzicz MA, Lustig LR, Usami SI, Boudewyns AN. Congenital hearing loss. Nat Rev Dis Primers. 2017;3:16094. - PMC - PubMed

[8] Korver AM, Smith RJ, Van Camp G, Schleiss MR, Bitner-Glindzicz MA, Lustig LR, Usami SI, Boudewyns AN. Congenital hearing loss. Nat Rev Dis Primers. 2017;3:16094. - PMC - PubMed

[9] Chan DK, Chang KW. GJB2-associated hearing loss: systematic review of worldwide prevalence, genotype, and auditory phenotype. Laryngoscope. 2014;124(2):E34–53. - PubMed

[10] Chan DK, Chang KW. GJB2-associated hearing loss: systematic review of worldwide prevalence, genotype, and auditory phenotype. Laryngoscope. 2014;124(2):E34–53. - PubMed

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The pathogenesis of common Gjb2 mutations associated with human hereditary deafness in mice

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The pathogenesis of common Gjb2 mutations associated with human hereditary deafness in mice

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