Image_1_Hearing Loss in Id1−/−; Id3+/− and Id1+/−; Id3−/− Mice Is Associated With a High Incidence of Middle Ear Infection (Otitis Media).TIF

Inhibitors of differentiation/DNA binding (Id) proteins are crucial for inner ear development, but whether Id mutations affect middle ear function remains unknown. In this study, we obtained Id1−/−; Id3+/− mice and Id1+/−; Id3−/− mice and carefully examined their middle ear morphology and auditory function. Our study revealed a high incidence (>50%) of middle ear infection in the compound mutant mice. These mutant mice demonstrated hearing impairment starting around 30 days of age, as the mutant mice presented elevated auditory brainstem response (ABR) thresholds compared to those of the littermate controls. The distortion product of otoacoustic emission (DPOAE) was also used to evaluate the conductive function of the middle ear, and we found much lower DPOAE amplitudes in the mutant mice, suggesting sound transduction in the mutant middle ear is compromised. This is the first study of the middle ears of Id compound mutant mice, and high incidence of middle ear infection determined by otoscopy and histological analysis of middle ear suggests that Id1/Id3 compound mutant mice are a novel model for human otitis media (OM).</p

ABR and DPOAE analysis in Atoh1-LKB1^-/- and wild-type mice at P30.

<p>(A, B) ABR measurements for broadband click (A) and frequency-specific pure tone stimulation (B) of Atoh1-LKB1^-/- mice and wild-type mice at P30. (C) DPOAE measurements of Atoh1-LKB1^-/- mice and wild-type mice at P30. ABR and DPOAE thresholds in Atoh1-LKB1^-/- mice were significantly higher than those in controls. *p<0.05; **p<0.01; ***p<0.001 compared with WT threshold at the corresponding frequency as determined by Student’s t-test; n>4 animals for each experiment and genotype.</p

Generation of Atoh1-LKB1^-/- conditional knockout mice.

<p>(A) Mouse genotyping by PCR analysis. Lanes: wild-type (+/+), heterozygous (+/-) and homozygous (-/-) mice. The LKB1 gene product of the wild-type allele was 415-bp. A 415-bp band and an 800-bp band were detected in the heterozygous mice. Only the 800-bp band was observed in the homozygous mice. The PCR product of the Atoh1-Cre recombinase gene was 319-bp. (B) Western blot analysis of Lkb1 in the cochlea of Atoh1-LKB1^-/- and wild-type mice at P14. A 48.6-kD LKB1 protein was decreased obviously in the cochlea of Atoh1-LKB1^-/- mice compared with the controls. (C) Gross morphology of wild-type and Atoh1-LKB1^-/- mice at P21. There was no obvious difference, except for the smaller size of the mutant mice compared with the controls. (D) The body weight of male and female mice from WT and mutant mice was measured. The mutant mice exhibited significant decreases in weight compared to those of the control group after P21. Error bars indicate SEM. *p<0.05; **p<0.01; ***p<0.001 by Student’s t-test. n = 5 mice per experiment and genotype.</p

Model of the function of LKB1 in hair cells of the inner ear.

<p>LKB1 regulates the development and maintenance of hair cell stereociliary bundles in the inner ear by the activation of ERM. Strad and Mo25 interact and activate LKB1, which may regulate the level of phosphorylated and activated ERM proteins. The phosphorylated ERM protein (p-ERM) controls the actin assembly in the development and maintenance of stereocilia. Deletion of LKB1 in our mutant mice caused a significantly decreased level of pERM, which resulted in abnormal actin assembly. The abnormal development of stereocilia caused hair cell loss and hearing impairment in LKB1-deficienct mice.</p

The stereocilia abnormalities of cochlea OHCs in Atoh1-LKB1^-/- mice.

<p>(A) There was no significant difference in temporal bones between mutant and wild-type mice at P14. (B) Hematoxylin and eosin (HE) staining showed no obvious abnormal morphology of the organ of Corti in Atoh1-LKB1^-/- mice. Scale bar: 100 μm. (C) Confocal images of cochlear whole mounts stained with phalloidin. At P1, the hair cell stereocilia showed minor abnormalities in mutant OHCs. The disorder of stereociliary bundles of outer hair cells began to develop and the stereociliary bundle displayed a “U” shape instead of “V” shape in Atoh1-LKB1^-/- mice at P7(white arrow), and the phenotypes became worse at P21 (white arrow). Consistent with the sporadic loss of pillar cell (arrowhead), prominent loss of outer hair cells (asterisk) was also observed in Atoh1-LKB1^-/- mice at P21. Green, phalloidin, an F-actin specific dye. Scale bars: 20 μm. (D) Scanning electron micrograph (SEM) of hair cell stereocilia in the middle-basal turn of the Atoh1-LKB1^-/- and control cochlea at P21. Top panel: Stereociliary bundles of hair cells were abnormal (black arrows), and there was obvious loss of outer hair cells (asterisk) in the Atoh1-LKB1^-/- mice. Scale bars: 50 μm. Middle panel: amplified stereocilia of outer hair cells. Stereocilia of outer hair cells were degenerated and an abnormal “U” shape of the stereociliary bundle was displayed in Atoh1-LKB1^-/- mice (black arrows). Scale bars: 20 μm. Bottom panel: amplified stereocilia of inner hair cells. A minor disorder in stereociliary bundles was observed in Atoh1-LKB1^-/- mice. Scale bars: 20 μm. OHCs: outer hair cells; IHCs: inner hair cells; HCs: hair cells. (E) Scanning electron micrograph (SEM) of stereocilia of the middle-basal turn of outer hair cells in the high resolution. Stereociliary bundles showed degeneration (arrowhead) and an abnormal “U” shape in Atoh1-LKB1^-/- mice at P21. Scale bars: 20 μm. (F) Quantitative assessment of the apical surfaces area of outer hair cells in the middle turns of the cochlea at P21. The area of apical surfaces of the hair cells was significantly decreased in the mutant mice compared with that of the controls. Error bars indicate SEM. *p<0.05; **p<0.01; ***p<0.001 compared with the WT group by Student’s t-test; n = 6 hair cells per genotype.</p

Percentage of remaining hair cells quantified from three areas of 200μm length near the base in each row of HCs at P30.

<p>All data are mean form 6 ears from the three wild-type mice (WT) or three Atoh1-LKB1^-/- mice (CKO), respectively.</p><p>Percentage of remaining hair cells quantified from three areas of 200μm length near the base in each row of HCs at P30.</p

Localization of ERM and the decrease of pERM level in mutant cochlea.

<p>(A) Localization of ERM proteins in the hair cells. ERM staining was detected in the bundles of hair cells in both two types of mice at P14. Red, phalloidin; Green, ERM. Scale bars: 20 μm. (B, C) Western blot analysis of the cochlea of mice at P14. Total ERM proteins had a little increase (B), while the level of phosphorylated ERM (pERM) was significantly lower (C) in the Atoh1-LKB1^–/– mice (-/-) compared with the controls (+/+). Error bars indicate SEM. *p<0.05; **p<0.01; ***p<0.001 compared with WT by Student’s t-test; n≥4 animals per genotype. Scale bars: 20 μm.</p

The cell loss of cochlea OHCs in the Atoh1-LKB1^-/- mice.

<p>Confocal whole-mount images of cochlea in the control and LKB1 mutant cochlea at P21. Hair cells were labeled with the hair cell marker myosin VIIa (green) (A), outer hair cell lateral membrane marker Prestin (green) (B) and the cell nucleus-specific dye DAPI (blue) (C). The morphology of the hair cell cytoplasm, membrane, and nucleus looked normal in the mutant hair cells and controls except for the sporadic loss of outer hair cells. (D) Immunostaining of the cryosections of the cochlea from the midbasal region of control and LKB1 mutant mice at P21 by myosin VIIa (green) and PI (red). The images showed that hair cells in the mutant mice exhibit normal morphology except for the hair cell loss (white arrows). Scale bars: 20 μm.</p

Image_1_SMPX Deficiency Causes Stereocilia Degeneration and Progressive Hearing Loss in CBA/CaJ Mice.JPEG

The small muscle protein, x-linked (SMPX) encodes a small protein containing 88 amino acids. Malfunction of this protein can cause a sex-linked non-syndromic hearing loss, named X-linked deafness 4 (DFNX4). Herein, we reported a point mutation and a frameshift mutation in two Chinese families who developed gradual hearing loss with age. To explore the impaired sites in the hearing system and the mechanism of DFNX4, we established and validated an Smpx null mouse model using CRISPR-Cas9. By analyzing auditory brainstem response (ABR), male Smpx null mice showed a progressive hearing loss starting from high frequency at the 3rd month. Hearing loss in female mice was milder and occurred later compared to male mice, which was very similar to human beings. Through morphological analyses of mice cochleas, we found the hair cell bundles progressively degenerated from the shortest row. Cellular edema occurred at the end phase of stereocilia degeneration, followed by cell death. By transfecting exogenous fluorescent Smpx into living hair cells, Smpx was observed to be expressed in stereocilia. Through noise exposure, it was shown that Smpx might participate in maintaining hair cell bundles. This Smpx knock-out mouse might be used as a suitable model to explore the pathology of DFNX4.</p

Table_1_SMPX Deficiency Causes Stereocilia Degeneration and Progressive Hearing Loss in CBA/CaJ Mice.DOC

The small muscle protein, x-linked (SMPX) encodes a small protein containing 88 amino acids. Malfunction of this protein can cause a sex-linked non-syndromic hearing loss, named X-linked deafness 4 (DFNX4). Herein, we reported a point mutation and a frameshift mutation in two Chinese families who developed gradual hearing loss with age. To explore the impaired sites in the hearing system and the mechanism of DFNX4, we established and validated an Smpx null mouse model using CRISPR-Cas9. By analyzing auditory brainstem response (ABR), male Smpx null mice showed a progressive hearing loss starting from high frequency at the 3rd month. Hearing loss in female mice was milder and occurred later compared to male mice, which was very similar to human beings. Through morphological analyses of mice cochleas, we found the hair cell bundles progressively degenerated from the shortest row. Cellular edema occurred at the end phase of stereocilia degeneration, followed by cell death. By transfecting exogenous fluorescent Smpx into living hair cells, Smpx was observed to be expressed in stereocilia. Through noise exposure, it was shown that Smpx might participate in maintaining hair cell bundles. This Smpx knock-out mouse might be used as a suitable model to explore the pathology of DFNX4.</p