Spatiotemporal expression of TRPM4 in the mouse cochlea

The present study was conducted to elucidate the presence of the transient receptor potential cation channel subfamily M member 4, TRPM4, in the mouse inner ear. TRPM4 immunoreactivity (IR) was found in the cell body of inner hair cells (IHCs) in the organ of Corti in the apical side of marginal cells of the stria vascularis, in the apical portion of the dark cells of the vestibule, and in a subset of the type II neurons in the spiral ganglion. Subsequently, changes in the distribution and expression of TRPM4 in the inner ear during embryonic and postnatal developments were also evaluated. Immunohistochemical localization demonstrated that the emergence of the TRPM4-IR in IHCs occurs shortly before the onset of hearing, whereas that in the marginal cells happens earlier, at the time of birth, coinciding with the onset of endolymph formation. Furthermore, semiquantitative real-time PCR assay showed that expressions of TRPM4 in the organ of Corti and in the stria vascularis increased dramatically at the onset of hearing. Because TRPM4 is a Ca2+-activated monovalent-selective cation channel, these findings imply that TRPM4 contributes to potassium ion transport, essential for the signal transduction in IHCs and the formation of endolymph by marginal cells. © 2014 Wiley Periodicals, Inc

An unusually powerful mode of low-frequency sound interference due to defective hair bundles of the auditory outer hair cells

International audienceA detrimental perceptive consequence of damaged auditory sen-sory hair cells consists in a pronounced masking effect exerted by low-frequency sounds, thought to occur when auditory threshold elevation substantially exceeds 40 dB. Here, we identified the submembrane scaffold protein Nherf1 as a hair-bundle component of the differentiating outer hair cells (OHCs). Nherf1 −/− mice dis-played OHC hair-bundle shape anomalies in the mid and basal co-chlea, normally tuned to mid-and high-frequency tones, and mild (22–35 dB) hearing-threshold elevations restricted to midhigh sound frequencies. This mild decrease in hearing sensitivity was, however, discordant with almost nonresponding OHCs at the co-chlear base as assessed by distortion-product otoacoustic emissions and cochlear microphonic potentials. Moreover, unlike wild-type mice, responses of Nherf1 −/− mice to high-frequency (20–40 kHz

Analysis of subcellular localization of Myo7a, Pcdh15 and Sans in Ush1c knockout mice

Usher syndrome (USH) is the most frequent cause of combined deaf-blindness in man. An important finding from mouse models and molecular studies is that the USH proteins are integrated into a protein network that regulates inner ear morphogenesis. To understand further the function of harmonin in the pathogenesis of USH1, we have generated a targeted null mutation Ush1c mouse model. Here, we examine the effects of null mutation of the Ush1c gene on subcellular localization of Myo7a, Pcdh15 and Sans in the inner ear. Morphology and proteins distributions were analysed in cochlear sections and whole mount preparations from Ush1c(-/-) and Ush1c(-/+) controls mice. We observed the same distribution of Myo7a throughout the cytoplasm in knockout and control mice. However, we detected Pcdh15 at the base of stereocilia and in the cuticular plate in cochlear hair cells from Ush1c(+/-) controls, whereas in the knockout Ush1c(-/-) mice, Pcdh15 staining was concentrated in the apical region of the outer hair cells and no defined staining was detected at the base of stereocilia nor in the cuticular plate. We showed localization of Sans in the stereocilia of controls mouse cochlear hair cells. However, in cochleae from Ush1c(-/-) mice, strong Sans signals were detected towards the base of stereocilia close to their insertion point into the cuticular plate. Our data indicate that the disassembly of the USH1 network caused by absence of harmonin may have led to the mis-localization of the Protocadherin 15 and Sans proteins in the cochlear hair cells of Ush1c(-/-) knockout mice

Deformation of the Outer Hair Cells and the Accumulation of Caveolin-2 in Connexin 26 Deficient Mice.

Mutations in GJB2, which encodes connexin 26 (Cx26), a cochlear gap junction protein, represent a major cause of pre-lingual, non-syndromic deafness. The degeneration of the organ of Corti observed in Cx26 mutant-associated deafness is thought to be a secondary pathology of hearing loss. Here we focused on abnormal development of the organ of Corti followed by degeneration including outer hair cell (OHC) loss.We investigated the crucial factors involved in late-onset degeneration and loss of OHC by ultrastructural observation, immunohistochemistry and protein analysis in our Cx26-deficient mice (Cx26f/fP0Cre).In ultrastructural observations of Cx26f/fP0Cre mice, OHCs changed shape irregularly, and several folds or notches were observed in the plasma membrane. Furthermore, the mutant OHCs had a flat surface compared with the characteristic wavy surface structure of OHCs of normal mice. Protein analysis revealed an increased protein level of caveolin-2 (CAV2) in Cx26f/fP0Cre mouse cochlea. In immunohistochemistry, a remarkable accumulation of CAV2 was observed in Cx26f/fP0Cre mice. In particular, this accumulation of CAV2 was mainly observed around OHCs, and furthermore this accumulation was observed around the shrunken site of OHCs with an abnormal hourglass-like shape.The deformation of OHCs and the accumulation of CAV2 in the organ of Corti may play a crucial role in the progression of, or secondary OHC loss in, GJB2-associated deafness. Investigation of these molecular pathways, including those involving CAV2, may contribute to the elucidation of a new pathogenic mechanism of GJB2-associated deafness and identify effective targets for new therapies

GJPs were significantly shorter in Brn4-deficient mice as compared with littermate controls at 6 weeks.

<p>The lengths of the largest GJPs (e.g., brackets in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108216#pone-0108216-g001" target="_blank">Figure 1 K and L</a>) along a single cell border were measured in the <i>z</i>-stack confocal images and are expressed as the mean ± Standard Error (SE) (<i>n</i> = 81 GJPs per mouse type). ***<i>P</i> = 6.49×10^–16 (Student’s <i>t</i>-test).</p

Deficiency of Transcription Factor Brn4 Disrupts Cochlear Gap Junction Plaques in a Model of DFN3 Non-Syndromic Deafness

<div><p><i>Brn4</i>, which encodes a POU transcription factor, is the gene responsible for DFN3, an X chromosome–linked, non-syndromic type of hearing loss. Brn4-deficient mice have a low endocochlear potential (EP), hearing loss, and ultrastructural alterations in spiral ligament fibrocytes, however the molecular pathology through which Brn4 deficiency causes low EP is still unclear. Mutations in the Gjb2 and Gjb6 genes encoding the gap junction proteins connexin26 (Cx26) and connexin30 (Cx30) genes, respectively, which encode gap junction proteins and are expressed in cochlear fibrocytes and non-sensory epithelial cells (i.e., cochlear supporting cells) to maintain the proper EP, are responsible for hereditary sensorineural deafness. It has been hypothesized that the gap junction in the cochlea provides an intercellular passage by which K⁺ is transported to maintain the EP at the high level necessary for sensory hair cell excitation. Here we analyzed the formation of gap junction plaques in cochlear supporting cells of Brn4-deficient mice at different stages by confocal microscopy and three-dimensional graphic reconstructions. Gap junctions from control mice, which are composed mainly of Cx26 and Cx30, formed linear plaques along the cell-cell junction sites with adjacent cells. These plaques formed pentagonal or hexagonal outlines of the normal inner sulcus cells and border cells. Gap junction plaques in Brn4-deficient mice did not, however, show the normal linear structure but instead formed small spots around the cell-cell junction sites. Gap junction lengths were significantly shorter, and the level of Cx26 and Cx30 was significantly reduced in Brn4-deficient mice compared with littermate controls. Thus the Brn4 mutation affected the assembly and localization of gap junction proteins at the cell borders of cochlear supporting cells, suggesting that Brn4 substantially contributes to cochlear gap junction properties to maintain the proper EP in cochleae, similar to connexin-related deafness.</p></div

Gap junction protein levels were reduced in Brn4-deficient mice at 6 weeks.

<p>Western blot analysis of Cx26 and Cx30 was performed in 6-week-old Brn4-deficient mice and littermate controls. The level of Cx26 and Cx30 was normalized to the corresponding β-actin levels and expressed relative to the amount present in each of the littermate controls. Values represent the mean ± SE (<i>n</i> = 3 mice). ***<i>P</i> = 7.943×10^–3 for Cx26 and ***<i>P</i> = 8.137×10^–3 for Cx30, Student’s <i>t</i>-test.</p

Brn4-deficient mice show a drastic disruption of GJPs with increasing age.

<p>Z-Stack images of cochlear inner sulcus cells from 2-, 4-, and 6-week-old littermate control and Brn4-deficient mice were obtained by confocal microscopy. GJPs were immunolabeled for Cx30 (red) (A–L). Nuclei were counterstained with DAPI (blue) (A–L). The positions of inner hair cells (IHCs) indicate the orientation of the images (A, B, E, F, I and J). High-magnification images of each boxed region in A, B, E, F, I and J are shown in C, D, G, H, K and L. The GJPs in 6-week-old Brn4-deficient mice were present as small spots around the cell-cell junction sites (J, L). In 2-week-old and 4-week-old littermate control and Brn4-deficient mice, the linear structure of the GJPs was present, although some plaques 4-week-old Brn4-deficient mice were disrupted and scattered (H, arrowheads). The lengths of the largest GJPs (brackets in K and L) along a single cell border were analyzed and are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108216#pone-0108216-g004" target="_blank">Figure 4</a>. Scale bars indicate 10 µm.</p