A connexin30 mutation rescues hearing and reveals roles for gap junctions in cochlear amplification and micromechanics.

Accelerated age-related hearing loss disrupts high-frequency hearing in inbred CD-1 mice. The p.Ala88Val (A88V) mutation in the gene coding for the gap-junction protein connexin30 (Cx30) protects the cochlear basal turn of adult CD-1Cx30A88V/A88V mice from degeneration and rescues hearing. Here we report that the passive compliance of the cochlear partition and active frequency tuning of the basilar membrane are enhanced in the cochleae of CD-1Cx30A88V/A88V compared to CBA/J mice with sensitive high-frequency hearing, suggesting that gap junctions contribute to passive cochlear mechanics and energy distribution in the active cochlea. Surprisingly, the endocochlear potential that drives mechanoelectrical transduction currents in outer hair cells and hence cochlear amplification is greatly reduced in CD-1Cx30A88V/A88V mice. Yet, the saturating amplitudes of cochlear microphonic potentials in CD-1Cx30A88V/A88V and CBA/J mice are comparable. Although not conclusive, these results are compatible with the proposal that transmembrane potentials, determined mainly by extracellular potentials, drive somatic electromotility of outer hair cells.

Evaluation of an independent prestin mouse model derived from the 129S1 strain.

Studies using the prestin knockout mouse indicate that removal of the outer hair cell (OHC) motor protein is associated with loss of sensitivity, frequency selectivity and somatic electromotility. Here we provide data obtained from another prestin mouse model that was produced commercially. In vivo electrical recordings from the round window indicate that the phenotype is similar to that of the original knockout generated by the Zuo group at St. Jude Children's Research Hospital. Hence, compound action potential (CAP) thresholds are shifted in a frequency-dependent manner and CAP tuning curves at 12 kHz are flat for masker frequencies between 3 and 18 kHz. Although CAP input-output functions at 6 kHz show a shift in sensitivity at low levels, responses approach wild-type magnitudes at high levels where the cochlear amplifier has less influence. In order to confirm that the loss of sensitivity and frequency selectivity is due to loss of prestin, we performed immunohistochemistry using a prestin antibody. Cochlear segments from homozygous mutant mice showed no fluorescence, while wild-type mice displayed a fluorescent signal targeted to the OHC's lateral membrane. Absence of prestin protein was confirmed using LDS-PAGE/Western blot analysis. These results indicate that the loss of function phenotype is associated with loss of prestin protein. Lack of prestin protein also results in a shortening of OHC length to approximately 60% of wild-type, similar to that reported previously by Liberman's group. The linkage shown between the loss of prestin protein and abnormal cochlear function validates the original knockout and attests to the importance of OHC motor function in the auditory periphery.

Connexin30 deficiency causes instrastrial fluid-blood barrier disruption within the cochlear stria vascularis.

The endocochlear potential (EP) is essential to hearing, because it provides approximately half of the driving force for the mechanoelectrical transduction current in auditory hair cells. The EP is produced by the stria vascularis (SV), a vascularized bilayer epithelium of the cochlea lateral wall. The absence of the gap junction protein connexin30 (Cx30) in Cx30(-/-) mice results in the SV failure to produce an EP, which mainly accounts for the severe congenital hearing impairment of these mice. Here, we show that the SV components of the EP electrogenic machinery and the epithelial barriers limiting the intrastrial fluid space, which are both necessary for the EP production, were preserved in Cx30(-/-) mice. In contrast, the endothelial barrier of the capillaries supplying the SV was disrupted before EP onset. This disruption is expected to result in an intrastrial electric shunt that is sufficient to account for the absence of the EP production. Immunofluorescence analysis of wild-type mice detected Cx30 in the basal and intermediate cells of the SV but not in the endothelial cells of the SV capillaries. Moreover, dye-coupling experiments showed that endothelial cells were not coupled to the SV basal, intermediate, and marginal cells. SV transcriptome analysis revealed a significant down-regulation of betaine homocysteine S-methyltransferase (Bhmt) in the Cx30(-/-) mice, which was restricted to the SV and resulted in a local increase in homocysteine, a known factor of endothelial dysfunction. Disruption of the SV endothelial barrier is a previously undescribed pathogenic process underlying hearing impairment.

GATA3 haploinsufficiency causes a rapid deterioration of distortion product otoacoustic emissions (DPOAEs) in mice.

Human HDR (hypoparathyroidism, deafness and renal dysplasia)-syndrome is caused by haploinsufficiency of zinc-finger transcription factor GATA3. The hearing loss due to GATA3 haploinsufficiency has been shown to be peripheral in origin, but it is unclear to what extent potential aberrations in the outer hair cells (OHCs) contribute to this disorder. To further elucidate the pathophysiological mechanism underlying the hearing defect in HDR-syndrome, we investigated the OHCs in heterozygous Gata3-knockout mice at both the functional and morphological level. While the signal-to-noise ratios of distortion product otoacoustic emissions (DPOAE) in wild type mice did not change significantly during the first half-year of live, those in the heterozygous Gata3 mice decreased dramatically. In addition, both light microscopic and transmission electron microscopic analyses showed that the number of OHCs containing vacuoles was increased in the mutants. Together, these findings indicate that outer hair cell malfunctioning plays a major role in the hearing loss in HDR-syndrome.

A targeted deletion in alpha-tectorin reveals that the tectorial membrane is required for the gain and timing of cochlear feedback.

alpha-tectorin is an extracellular matrix molecule of the inner ear. Mice homozygous for a targeted deletion in a-tectorin have tectorial membranes that are detached from the cochlear epithelium and lack all noncollagenous matrix, but the architecture of the organ of Corti is otherwise normal. The basilar membranes of wild-type and alpha-tectorin mutant mice are tuned, but the alpha-tectorin mutants are 35 dB less sensitive. Basilar membrane responses of wild-type mice exhibit a second resonance, indicating that the tectorial membrane provides an inertial mass against which outer hair cells can exert forces. Cochlear microphonics recorded in alpha-tectorin mutants differ in both phase and symmetry relative to those of wild-type mice. Thus, the tectorial membrane ensures that outer hair cells can effectively respond to basilar membrane motion and that feedback is delivered with the appropriate gain and timing required for amplification.

Combined cochleo-saccular and neuroepithelial abnormalities in the Varitint-waddler-J (VaJ) mouse.

Hearing loss in Varitint-waddler-J (VaJ) mice is of mixed origin with both cochleo-saccular and neuroepithelial components. Both VaJ/VaJ and VaJ/+ mutants show impaired cochlear function, but the homozygotes are more severely affected than heterozygotes. Neither group have any detectable compound action potential. Cochlear microphonics are only seen in half of the heterozygotes, at a reduced amplitude and raised threshold, and are not detected in any homozygotes. Summating potentials (SP) responses are seen in most of the heterozygotes, at high stimulus levels. The only responses in homozygotes were negative SPs seen in half of the mutants at very high sound levels, while the remaining homozygotes showed no responses to sound stimulation. Endocochlear potentials (EP) were often small or absent in both groups of mutants, with the homozygotes being more severely affected. Reduced pigmentation in the stria vascularis appears to be associated with a reduced EP, while a primary defect of the neuroepithelium, detectable by electron microscopy in hair cells of 14 day old mice, dramatically influences evoked potentials.

Additional findings on heritability and prenatal masculinization of cochlear mechanisms: click-evoked otoacoustic emissions.

A previous demonstration of a substantial genetic contribution to the expression of spontaneous otoacoustic emissions (SOAEs) is here extended to an aspect of click-evoked otoacoustic emissions (CEOAEs). CEOAEs were measured in the same twins and non-twins used for the SOAE heritability study. The stimuli were 100-microsecond clicks presented a nominal rate of 2/s; the emitted waveforms from 50 clicks were summed, and a 20-ms sample of that averaged waveform (beginning 6 ms after click presentation) was subjected to spectral analysis. The total power in the spectrum from 1 to 5 kHz in this temporal segment of the CEOAE waveform was used as the primary dependent variable. This overall power was significantly greater in female and right ears than in male and left ears, but the difference between dark- and light-eyed subjects was not significant. The overall power in the two left, and two right, ears of monozygotic co-twins was more highly correlated than in dizygotic co-twins, and structural modeling indicated that about 65-85% of the individual variation in the expression of CEOAE power could be attributed to genes-essentially the same heritability estimate as obtained previously from the SOAE data. Within-subject correlations between CEOAE power and number of SOAEs ranged from about 0.3 to 0.7, suggesting that these two forms of otoacoustic emission may depend upon somewhat different aspects of the same underlying mechanism and, thus, that heritability estimates based on one measure are not completely redundant to those from the other. While the average spectral power of the CEOAEs in opposite-sex dizygotic (OSDZ) females was smaller than that in same-sex dizygotic (SSDZ) females- and thus approached the value for males-the difference did not achieve statistical significance. Thus, the evidence for a prenatal masculinizing effect was less definitive in these CEOAE data than in the SOAE data obtained from the same subjects. An interpretation that accounts for both the CEOAE and SOAE results is that the strength of the so-called cochlear amplifiers is under genetic control that is to some extent mediated and/or modified through prenatal exposure to androgens. The indicated direction of effect is that weak cochlear amplifiers result when prenatal androgen levels are high. Under this view, then, androgen level contribute both to the sex differences observed in otoacoustic emissions and the prenatal masculinizing effects observed in opposite-sex twins, and they may be a factor in individual differences in OAE expression as well. Additionally it is shown that, although the powers of the CEOAE waveforms were reasonably highly correlated for the two ears of subjects in all groups, and across MZ co-twins, cross-correlations on the fine structures of those same pairs of CEOAE waveforms were essentially zero-presumably owing largely to the synchronizing of (different) SOAE frequencies in the ears being compared.