Localization and functional studies of pendrin in the mouse inner ear provide insight about the etiology of deafness in pendred syndrome.
ABSTRACT: Immunolocalization studies of mouse cochlea and vestibular end-organ were performed to study the expression pattern of pendrin, the protein encoded by the Pendred syndrome gene (PDS), in the inner ear. The protein was restricted to the areas composed of specialized epithelial cells thought to play a key role in regulating the composition and resorption of endolymph. In the cochlea, pendrin was abundant in the apical membrane of cells in the spiral prominence and outer sulcus cells (along with their root processes). In the vestibular end-organ, pendrin was found in the transitional cells of the cristae ampullaris, utriculi, and sacculi as well as in the apical membrane of cells in the endolymphatic sac. Pds-knockout (Pds-/-) mice were found to lack pendrin immunoreactivity in all of these locations. Histological studies revealed that the stria vascularis in Pds-/- mice was only two-thirds the thickness seen in wild-type mice, with the strial marginal cells showing irregular shapes and sizes. Functional studies were also performed to examine the role of pendrin in endolymph homeostasis. Using double-barreled electrodes placed in both the cochlea and the utricle, the endocochlear potential and endolymph potassium concentration were measured in wild-type and Pds-/- mice. Consistent with the altered strial morphology, the endocochlear potential in Pds-/- mice was near zero and did not change during anoxia. On the other hand, the endolymphatic potassium concentration in Pds-/- mice was near normal in the cochlea and utricle. Together, these results suggest that pendrin serves a key role in the functioning of the basal and/or intermediate cells of the stria vascularis to maintain the endocochlear potential, but not in the potassium secretory function of the marginal cells.
Project description:Pendrin mutations cause enlarged vestibular aqueducts and various degrees of sensorineural hearing loss. The selective abolition of pendrin causes dilation of the membranous labyrinth known as endolymphatic hydrops, loss of the endocochlear potential, and consequently loss of hearing function. Because Na+ transport is one of the most important driving forces for fluid transport, the epithelial Na+ channel (ENaC) is believed to play an important role in fluid volume regulation in the inner ear. Therefore, the dysfunction of Na+ transport through ENaC by the acidification of endolymph in Pendred syndrome is one of the potential causes of endolymphatic hydrops. We investigated the changes of ENaC expression and function during the development of the pendrin knock-out mouse. In the cochlea, the expression of ? and ?ENaC was significantly increased at P56 in Pds-/- mice compared with Pds+/+ mice. In the vestibule, the expression of ?ENaC was significantly increased at P56, and ?ENaC expression significantly increased from P6 to P56 in Pds-/- mice. The ENaC-dependent trans-epithelial current was not significantly different between Pds+/+ and Pds-/- mice in Reissner's membrane or the saccular extramacular roof epithelium at P0, but the current was significantly increased in Pds-/- mice at P56 compared with Pds+/+ mice. These findings indicate that the expression and function of ENaC were enhanced in Pds-/- mice after the development of endolymphatic hydrops as a compensatory mechanism. This result provides insight into the role of Na+ transport in the development and regulation of endolymphatic hydrops due to pendrin mutations.
Project description:Mutations of SLC26A4 are a common cause of human hearing loss associated with enlargement of the vestibular aqueduct. SLC26A4 encodes pendrin, an anion exchanger expressed in a variety of epithelial cells in the cochlea, the vestibular labyrinth and the endolymphatic sac. Slc26a4 (?/?) mice are devoid of pendrin and develop a severe enlargement of the membranous labyrinth, fail to acquire hearing and balance, and thereby provide a model for the human phenotype. Here, we generated a transgenic mouse line that expresses human SLC26A4 controlled by the promoter of ATP6V1B1. Crossing this transgene into the Slc26a4 (?/?) line restored protein expression of pendrin in the endolymphatic sac without inducing detectable expression in the cochlea or the vestibular sensory organs. The transgene prevented abnormal enlargement of the membranous labyrinth, restored a normal endocochlear potential, normal pH gradients between endolymph and perilymph in the cochlea, normal otoconia formation in the vestibular labyrinth and normal sensory functions of hearing and balance. Our study demonstrates that restoration of pendrin to the endolymphatic sac is sufficient to restore normal inner ear function. This finding in conjunction with our previous report that pendrin expression is required for embryonic development but not for the maintenance of hearing opens the prospect that a spatially and temporally limited therapy will restore normal hearing in human patients carrying a variety of mutations of SLC26A4.
Project description:Pendred's syndrome is an autosomal-recessive disorder characterized by deafness and goiter. After our recent identification of the human gene mutated in Pendred's syndrome (PDS), we sought to investigate in greater detail the expression of the gene and the function of its encoded protein (pendrin). Toward that end, we isolated the corresponding mouse ortholog (Pds) and performed RNA in situ hybridization on mouse inner ears (from 8 days postcoitum to postnatal day 5) to establish the expression pattern of Pds in the developing auditory and vestibular systems. Pds expression was detected throughout the endolymphatic duct and sac, in distinct areas of the utricle and saccule, and in the external sulcus region within the cochlea. This highly discrete expression pattern is unlike that of any other known gene and involves several regions thought to be important for endolymphatic fluid resorption in the inner ear, consistent with the putative functioning of pendrin as an anion transporter. These studies provide key first steps toward defining the precise role of pendrin in inner ear development and elucidating the pathogenic mechanism for the deafness seen in Pendred's syndrome.
Project description:In the cochlea, mammals maintain a uniquely high endolymphatic potential (EP), which is not observed in other vertebrate groups. However, a high [K(+)] is always present in the inner ear endolymph. Here, we show that Kir4.1, which is required in the mammalian stria vascularis to generate the highly positive EP, is absent in the functionally equivalent avian tegmentum vasculosum. In contrast, the molecular repertoire required for K(+) secretion, specifically NKCC1, KCNQ1, KCNE1, BSND and CLC-K, is shared between the tegmentum vasculosum, the vestibular dark cells and the marginal cells of the stria vascularis. We further show that in barn owls, the tegmentum vasculosum is enlarged and a higher EP (~+34 mV) maintained, compared to other birds. Our data suggest that both the tegmentum vasculosum and the stratified stria vascularis evolved from an ancestral vestibular epithelium that already featured the major cell types of the auditory epithelia. Genetic recruitment of Kir4.1 specifically to strial melanocytes was then a crucial step in mammalian evolution enabling an increase in the cochlear EP. An increased EP may be related to high-frequency hearing, as this is a hallmark of barn owls among birds and mammals among amniotes.
Project description:In the cochlea, K(+) is essential for mechano-electrical transduction. Here, we explore cochlear structure and function in mice lacking K(+) channels of the two-pore domain family. A profound deafness associated with a decrease in endocochlear potential is found in adult Kcnk5(-/-) mice. Hearing occurs around postnatal day 19 (P19), and completely disappears 2 days later. At P19, Kcnk5(-/-) mice have a normal endolymphatic [K(+)] but a partly lowered endocochlear potential. Using Lac-Z as a gene reporter, KCNK5 is mainly found in outer sulcus Claudius', Boettcher's and root cells. Low levels of expression are also seen in the spiral ganglion, Reissner's membrane and stria vascularis. Essential channels (KCNJ10 and KCNQ1) contributing to K(+) secretion in stria vascularis have normal expression in Kcnk5(-/-) mice. Thus, KCNK5 channels are indispensable for the maintenance of hearing. Among several plausible mechanisms, we emphasize their role in K(+) recycling along the outer sulcus lateral route.
Project description:Aminoglycosides enter inner ear hair cells across their apical membranes via endocytosis, or through the mechanoelectrical transduction channels in vitro, suggesting that these drugs enter cochlear hair cells from endolymph to exert their cytotoxic effect. We used zebrafish to determine if fluorescently tagged gentamicin (GTTR) also enters hair cells via apically located calcium-sensitive cation channels and the cytotoxicity of GTTR to hair cells. We then examined the serum kinetics of GTTR following systemic injection in mice and which murine cochlear sites preferentially loaded with systemically administered GTTR over time by confocal microscopy. GTTR is taken up by, and is toxic to, wild-type zebrafish neuromast hair cells. Neuromast hair cell uptake of GTTR is attenuated by high concentrations of extracellular calcium or unconjugated gentamicin and is blocked in mariner mutant zebrafish, suggestive of entry via the apical mechanotransduction channel. In murine cochleae, GTTR is preferentially taken up by the stria vascularis compared to the spiral ligament, peaking 3 h after intra-peritoneal injection, following GTTR kinetics in serum. Strial marginal cells display greater intensity of GTTR fluorescence compared to intermediate and basal cells. Immunofluorescent detection of gentamicin in the cochlea also revealed widespread cellular labeling throughout the cochlea, with preferential labeling of marginal cells. Only GTTR fluorescence displayed increasing cytoplasmic intensity with increasing concentration, unlike the cytoplasmic intensity of fluorescence from immunolabeled gentamicin. These data suggest that systemically administered aminoglycosides are trafficked from strial capillaries into marginal cells and clear into endolymph. If so, this will facilitate electrophoretically driven aminoglycoside entry into hair cells from endolymph. Trans-strial trafficking of aminoglycosides from strial capillaries to marginal cells will be dependent on as-yet-unidentified mechanisms that convey these drugs across the intra-strial electrical barrier and into marginal cells.
Project description:Mutations in human SLC26A4 are a common cause of hearing loss associated with enlarged vestibular aqueducts (EVA). SLC26A4 encodes pendrin, an anion-base exchanger expressed in inner ear epithelial cells that secretes HCO3- into endolymph. Studies of Slc26a4-null mice indicate that pendrin is essential for inner ear development, but have not revealed whether pendrin is specifically necessary for homeostasis. Slc26a4-null mice are profoundly deaf, with severe inner ear malformations and degenerative changes that do not model the less severe human phenotype. Here, we describe studies in which we generated a binary transgenic mouse line in which Slc26a4 expression could be induced with doxycycline. The transgenes were crossed onto the Slc26a4-null background so that all functional pendrin was derived from the transgenes. Varying the temporal expression of Slc26a4 revealed that E16.5 to P2 was the critical interval in which pendrin was required for acquisition of normal hearing. Lack of pendrin during this period led to endolymphatic acidification, loss of the endocochlear potential, and failure to acquire normal hearing. Doxycycline initiation at E18.5 or discontinuation at E17.5 resulted in partial hearing loss approximating the human EVA auditory phenotype. These data collectively provide mechanistic insight into hearing loss caused by SLC26A4 mutations and establish a model for further studies of EVA-associated hearing loss.
Project description:The recessive mouse mutant headbobber (hb) displays the characteristic behavioural traits associated with vestibular defects including headbobbing, circling and deafness. This mutation was caused by the insertion of a transgene into distal chromosome 7 affecting expression of native genes. We show that the inner ear of hb/hb mutants lacks semicircular canals and cristae, and the saccule and utricle are fused together in a single utriculosaccular sac. Moreover, we detect severe abnormalities of the cochlear sensory hair cells, the stria vascularis looks severely disorganised, Reissner's membrane is collapsed and no endocochlear potential is detected. Myo7a and Kcnj10 expression analysis show a lack of the melanocyte-like intermediate cells in hb/hb stria vascularis, which can explain the absence of endocochlear potential. We use Trp2 as a marker of melanoblasts migrating from the neural crest at E12.5 and show that they do not interdigitate into the developing strial epithelium, associated with abnormal persistence of the basal lamina in the hb/hb cochlea. We perform array CGH, deep sequencing as well as an extensive expression analysis of candidate genes in the headbobber region of hb/hb and littermate controls, and conclude that the headbobber phenotype is caused by: 1) effect of a 648 kb deletion on distal Chr7, resulting in the loss of three protein coding genes (Gpr26, Cpmx2 and Chst15) with expression in the inner ear but unknown function; and 2) indirect, long range effect of the deletion on the expression of neighboring genes on Chr7, associated with downregulation of Hmx3, Hmx2 and Nkx1.2 homeobox transcription factors. Interestingly, deletions of the orthologous region in humans, affecting the same genes, have been reported in nineteen patients with common features including sensorineural hearing loss and vestibular problems. Therefore, we propose that headbobber is a useful model to gain insight into the mechanisms underlying deafness in human 10qter deletion syndrome.
Project description:The His723Arg (H723R) mutation in SLC26A4, encoding pendrin, is the most prevalent mutation in East Asia, resulting in DFNB4, an autosomal recessive type of genetic hearing loss. Although the main pathological mechanism of H723R was identified as a protein-folding defect in pendrin, there is still no curative treatment for associated hearing loss. Here, we show that H723R-pendrin expression and activity are rescued by activation of the chaperonin DNAJC14. In vitro, DNAJC14 was activated via Japanese encephalitis virus (JEV) inoculation, and toxin-attenuated JEV rescued the surface expression and anion exchange activity of H723R-pendrin. Human H723R-pendrin transgenic mice (hH723R Tg) were established in a mouse slc26a4 knockout background, in which only hH723R-pendrin was expressed in the inner ear (Pax2-Cre dependent) to mimic human DFNB4 pathology. Crossing hH723R Tg with DNAJC14-overexpressing mice resulted in reduced cochlear hydrops and more preserved outer hair cells in the cochlea compared to those in hH723R Tg mice. Furthermore, the stria vascularis and spiral ligament were thicker and KCNJ10 expression was increased with DNAJC14 overexpression; however, hearing function and enlarged endolymphatic hydrops were not recovered. These results indicate that DNAJC14 overexpression ameliorates the cochlear degeneration caused by misfolded pendrin and might be a potential therapeutic target for DFNB4.
Project description:The ganglioside GM3 synthase (SAT-I), encoded by a single-copy gene, is a primary glycosyltransferase for the synthesis of complex gangliosides. In SAT-I null mice, hearing ability, assessed by brainstem auditory-evoked potentials (BAEP), was impaired at the onset of hearing and had been completely lost by 17 days after birth (P17), showing a deformity in hair cells in the organ of Corti. By 2 months of age, the organ of Corti had selectively and completely disappeared without effect on balance or motor function or in the histology of vestibule. Interestingly, spatiotemporal changes in localization of individual gangliosides, including GM3 and GT1b, were observed during the postnatal development and maturation of the normal inner ear. GM3 expressed in almost all regions of cochlea at P3, but at the onset of hearing it distinctly localized in stria vascularis, spiral ganglion, and the organ of Corti. In addition, SAT-I null mice maintain the function of stria vascularis, because normal potassium concentration and endocochlear potential of endolymph were observed even when they lost the BAEP completely. Thus, the defect of hearing ability of SAT-I null mice could be attributed to the functional disorganization of the organ of Corti, and the expression of gangliosides, especially GM3, during the early part of the functional maturation of the cochlea could be essential for the acquisition and maintenance of hearing function.