A mouse model for human deafness DFNB22 reveals that hearing impairment is due to a loss of inner hair cell stimulation.

The gene causative for the human nonsyndromic recessive form of deafness DFNB22 encodes otoancorin, a 120-kDa inner ear-specific protein that is expressed on the surface of the spiral limbus in the cochlea. Gene targeting in ES cells was used to create an EGFP knock-in, otoancorin KO (Otoa(EGFP/EGFP)) mouse. In the Otoa(EGFP/EGFP) mouse, the tectorial membrane (TM), a ribbon-like strip of ECM that is normally anchored by one edge to the spiral limbus and lies over the organ of Corti, retains its general form, and remains in close proximity to the organ of Corti, but is detached from the limbal surface. Measurements of cochlear microphonic potentials, distortion product otoacoustic emissions, and basilar membrane motion indicate that the TM remains functionally attached to the electromotile, sensorimotor outer hair cells of the organ of Corti, and that the amplification and frequency tuning of the basilar membrane responses to sounds are almost normal. The compound action potential masker tuning curves, a measure of the tuning of the sensory inner hair cells, are also sharply tuned, but the thresholds of the compound action potentials, a measure of inner hair cell sensitivity, are significantly elevated. These results indicate that the hearing loss in patients with Otoa mutations is caused by a defect in inner hair cell stimulation, and reveal the limbal attachment of the TM plays a critical role in this process.

Audiometric characteristics of two Dutch families with non-ocular Stickler syndrome (COL11A2).

OBJECTIVE: To evaluate hearing impairment and cochlear function in non-ocular Stickler syndrome. STUDY DESIGN: Multifamily study. PATIENTS & METHODS: Ten patients from two different families with non-ocular Stickler syndrome (Stickler syndrome type 3) were included. Six members of the first family and four members of the second family participated in this study. Otorhinolaryngologic examinations were performed. Pure-tone and speech audiograms were obtained. Longitudinal analysis was performed. Psychophysical measurements, including loudness scaling, gap detection, difference limen for frequency and speech perception in noise were administered to assess cochlear function at a deeper level. RESULTS: Affected individuals in the first family were carriers of a heterozygous splice donor mutation in the COL11A2 gene. Affected individuals in the second family were carriers of a novel heterozygous missense mutation in COL11A2. Both families showed bilateral, non-progressive hearing impairment with childhood onset. The severity of the hearing impairment exhibited inter- and intrafamilial variability and was mostly mild to moderate. The results of the psychophysical measurements were similar to those previously published for DFNA8/12 (TECTA) and DFNA13 (COL11A2) patients and thus consistent with an intra-cochlear conductive hearing impairment. This is in line with the theory that mutations in COL11A2 affect tectorial membrane function. CONCLUSION: Hearing impairment in non-ocular Stickler syndrome is characterized by non-progressive hearing loss, present since childhood, and mostly mild to moderate in severity. Psychophysical measurements in non-ocular Stickler patients were suggestive of intra-cochlear conductive hearing impairment.

The anterior shear and distraction tests for craniocervical instability. An evaluation using magnetic resonance imaging.

Screening for integrity of the ligaments of the craniocervical complex has been suggested prior to the application of manual techniques to the upper cervical spine. However, most tests proposed lack validation limiting their usefulness clinically. This study examined the effect of the anterior shear test for the transverse ligament and the distraction test for the tectorial membrane in normal volunteers. MRI was performed in supine in neutral and end-range stress test positions in 16 individuals using proton density-weighted sequences and a standard head coil in a 3-T system. Measurements were made with respect to a strictly standardised protocol. The anterior shear test was assessed using changes in atlantodental interval and distance from the anterior arch of the atlas to the posterior aspect of the odontoid process. Distraction testing for the tectorial membrane was assessed by changes in basion-dental interval and by direct measurement of the tectorial membrane. Differences were compared using Wilcoxon Sign Rank tests or paired t-test depending upon each variables assessment of normality. Anterior shear testing resulted in a 0.41 mm mean increase in atlantodental interval (p = 0.03) and 0.35 mm mean increase in axial plane distance (p = 0.05). Distraction testing for the tectorial membrane resulted in a 0.64 mm increase in basion-dental interval (p < 0.01) and a 1.11 mm increase in direct ligament length measurement (p = 0.02). Reliability of measurements ranged from moderate to substantial. These results indicate that these tests produce a consistent direct effect on the transverse ligament and the tectorial membrane which is consistent with their theorised mechanism for clinical use.

Deficient forward transduction and enhanced reverse transduction in the alpha tectorin C1509G human hearing loss mutation.

Most forms of hearing loss are associated with loss of cochlear outer hair cells (OHCs). OHCs require the tectorial membrane (TM) for stereociliary bundle stimulation (forward transduction) and active feedback (reverse transduction). Alpha tectorin is a protein constituent of the TM and the C1509G mutation in alpha tectorin in humans results in autosomal dominant hearing loss. We engineered and validated this mutation in mice and found that the TM was shortened in heterozygous Tecta(C1509G/+) mice, reaching only the first row of OHCs. Thus, deficient forward transduction renders OHCs within the second and third rows non-functional, producing partial hearing loss. Surprisingly, both Tecta(C1509G/+) and Tecta(C1509G/C1509G) mice were found to have increased reverse transduction as assessed by sound- and electrically-evoked otoacoustic emissions. We show that an increase in prestin, a protein necessary for electromotility, in all three rows of OHCs underlies this phenomenon. This mouse model demonstrates a human hearing loss mutation in which OHC function is altered through a non-cell-autonomous variation in prestin.

Deafness in TRbeta mutants is caused by malformation of the tectorial membrane.

Thyroid hormone receptor beta (TRbeta) dysfunction leads to deafness in humans and mice. Deafness in TRbeta(-/-) mutant mice has been attributed to TRbeta-mediated control of voltage- and Ca(2+)-activated K(+) (BK) channel expression in inner hair cells (IHCs). However, normal hearing in young constitutive BKalpha(-/-) mutants contradicts this hypothesis. Here, we show that mice with hair cell-specific deletion of TRbeta after postnatal day 11 (P11) have a delay in BKalpha expression but normal hearing, indicating that the origin of hearing loss in TRbeta(-/-) mutant mice manifested before P11. Analyzing the phenotype of IHCs in constitutive TRbeta(-/-) mice, we found normal Ca(2+) current amplitudes, exocytosis, and shape of compound action potential waveforms. In contrast, reduced distortion product otoacoustic emissions and cochlear microphonics associated with an abnormal structure of the tectorial membrane and enhanced tectorin levels suggest that disturbed mechanical performance is the primary cause of deafness resulting from TRbeta deficiency.

Hearing molecules: contributions from genetic deafness.

Considerable progress has been made over the past decade identifying many genes associated with deafness. With the identification of these hereditary deafness genes and the proteins they encode, molecular elements of basic hearing mechanisms emerge. As functional studies of these molecular elements become available, we can put together the pieces of the puzzle and begin to reach an understanding of the molecular mechanisms of hearing. The goal of this review is to discuss studies over the past decade that address the function of the proteins implicated in genetic deafness and to place them in the context of basic molecular mechanisms in hearing. The first part of this review highlights structural and functional features of the cochlea and auditory nerve. This background will provide a context for the second part, which addresses the molecular mechanisms underlying cochlear function as elucidated by genetic causes of deafness.

Genes related to hearing disorders.

The inner ear is a highly specialized organ and the mechanisms of its function are complex and have not yet fully been elucidated. For example, there are questions such as how the stereocilia characteristics of hair cells are regularly arranged, how the reactions of stereocilia and ion channels of hair cells to sound are controlled, and how the ion environment is maintained in the internal ear. The mechanisms of inner ear function are being elucidated by analysis of human hereditary hearing disorders and genetic and molecular biological techniques using mouse hearing disorder models. Understanding of the mechanism of inner ear function provides important information for treatment of the inner ear. This review outlines several findings obtained from humans with hereditary hearing disorders and mouse hearing disorder models.

Targeted disruption of otog results in deafness and severe imbalance.

Genes specifically expressed in the inner ear are candidates to underlie hereditary nonsyndromic deafness. The gene Otog has been isolated from a mouse subtractive cDNA cochlear library. It encodes otogelin, an N-glycosylated protein that is present in the acellular membranes covering the six sensory epithelial patches of the inner ear: in the cochlea (the auditory sensory organ), the tectorial membrane (TM) over the organ of Corti; and in the vestibule (the balance sensory organ), the otoconial membranes over the utricular and saccular maculae as well as the cupulae over the cristae ampullares of the three semi-circular canals. These membranes are involved in the mechanotransduction process. Their movement, which is induced by sound in the cochlea or acceleration in the vestibule, results in the deflection of the stereocilia bundle at the apex of the sensory hair cells, which in turn opens the mechanotransduction channels located at the tip of the stereo-cilia. We sought to elucidate the role of otogelin in the auditory and vestibular functions by generating mice with a targeted disruption of Otog. In Otog-/- mice, both the vestibular and the auditory functions were impaired. Histological analysis of these mutants demonstrated that in the vestibule, otogelin is required for the anchoring of the otoconial membranes and cupulae to the neuroepithelia. In the cochlea, ultrastructural analysis of the TM indicated that otogelin is involved in the organization of its fibrillar network. Otogelin is likely to have a role in the resistance of this membrane to sound stimulation. These results support OTOG as a possible candidate gene for a human nonsyndromic form of deafness.

Hair cell regeneration and recovery of function in the avian auditory system.

Chickens were exposed to an intense pure tone that destroyed the hair cells and tectorial membrane in a crescent shaped patch along the abneural edge of the basilar papilla. During the following weeks, when the hair cells and tectorial membrane were regenerating, psychophysical and electrophysiological measures were obtained to assess the time course and degree of recovery. Immediately after the exposure, the behavioral thresholds were elevated 30-40 dB and auditory temporal integration was greatly reduced; however, both measures fully recovered by 28 days post-exposure. In addition, tone-on-tone masking patterns recovered to normal. Immediately after the exposure, the thresholds of single cochlear ganglion neurons were elevated more than 30 dB, tuning curves were broader than normal, two-tone rate suppression (TTRS) boundary slopes were shallower than normal and spontaneous activity was reduced. Threshold and spontaneous discharge rate fully recovered after the exposure. Tuning and TTRS also recovered significantly in most neurons; however, some units with characteristic frequencies (CFs) near the exposure frequency showed abnormal tuning and TTRS suppression. The regeneration of the hair cells and lower honeycomb layer of the tectorial membrane is associated with considerable recovery of function; however, the incomplete recovery of tuning and TTRS in some neurons may be linked to the incomplete regeneration of the tectorial membrane.

Incomplete recovery of chicken distortion product otoacoustic emissions following acoustic overstimulation.

Distortion product otoacoustic emissions (DPOAEs) were measured in chickens before and after exposure to a 525-Hz pure tone (120 dB SPL, 48 h). The exposure caused extensive hair cell loss and destroyed the tectorial membrane along the abneural edge of the basilar papilla in the low-to-mid-frequency region of the cochlea. Although the lesion was restricted, DPOAEs were greatly depressed at all frequencies immediately after the exposure. The high-frequency DPOAEs gradually recovered to preexposure values after the exposure; however, there was little or no improvement in DPOAEs at test frequencies equal to or slightly above the exposure frequency even after 16 weeks of recovery. By 28 days of recovery, the previously damaged region of the basilar papilla had been repopulated by hair cells and the lower honeycomb layer of the tectorial membrane had regenerated, but not the upper fibrous layer. The upper fibrous layer of the tectorial membrane was still missing after 16 weeks of recovery and the region of damage corresponded closely to the frequency regions where the DPOAEs were depressed.

Long-term effect of acoustic trauma on distortion product otoacoustic emissions in chickens.

Distortion product otoacoustic emissions (DPOAE), 2F1-F2, were measured in chickens before and after exposure to a 1.5-kHz pure tone presented at 120-dB sound-pressure level for 48 h. The low-level component of the DPOAE input/output function was shifted to the right at all frequencies immediately after the exposure with the greatest effect occurring at and above the exposure frequency. The slope of the high-level component of the input/output also increased significantly for test frequencies close to and above the exposure frequency so that the amplitude of the DPOAE was equal to or greater than normal at high stimulus levels. DPOAE obtained at the highest and lowest frequencies were almost normal after 8 weeks of recovery; however, the input/output functions near the exposure frequency showed almost no improvement over the 8-week recovery period. The lack of recovery could conceivably be due to residual damage to hair cells that survived the exposure or to incomplete regeneration of the tectorial membrane.

Transitory endolymph leakage induced hearing loss and tinnitus: depolarization, biphasic shortening and loss of electromotility of outer hair cells.

There are types of deafness and tinnitus in which ruptures or massive changes in the ionic permeability of the membranes lining the endolymphatic space [e.g., of the reticular lamina (RL)] are believed to allow potassium-rich endolymph to deluge the low [K+] perilymphatic fluid (e.g., in the small spaces of Nuel). This would result in a K+ intoxication of sensory and neural structures. Acute attacks of Ménière's disease have been suggested to be an important example for this event. The present study investigated the effects of transiently elevated [K+] due to the addition of artificial endolymph to the basolateral cell surface of outer hair cells (OHC) in replicating endolymph-induced K+ intoxication of the perilymph in the small spaces of Nuel. The influence of K+ intoxication of the basolateral OHC cell surface on the transduction was then examined. Intoxication resulted in an inhibition of the physiological repolarizing K+ efflux from hair cells. This induced unwanted depolarizations of the hair cells, interfering with mechanoelectrical transduction. A pathological longitudinal OHC shortening was also found, with subsequent compression of the organ of Corti possibly influencing the micromechanics of the mechanically active OHC. Both micromechanical and electrophysiological alterations are proposed to contribute to endolymph leakage induced attacks of deafness and possibly also to tinnitus. Moreover, repeated or long-lasting K+ intoxications of OHC resulted in a chronic and complete loss of OHC motility. This is suggested to be a pathophysiological basis in some patients with chronic hearing loss resulting from Ménière's syndrome.

Video-enhanced DIC images of the noise-damaged and regenerated chick tectorial membrane.

Exposure of the chick cochlea to acoustic overstimulation results in a loss of hair cells and a disruption of the tectorial membrane. With time, new hair cells are produced to replace those that are lost and, concurrently, a new tectorial membrane is regenerated. Previous studies of tectorial membrane regeneration examined tissues that were fixed and processed for scanning and transmission electron microscopy. This processing results in a considerable shrinkage of the membrane, and, therefore, it was unclear how the noise damage and subsequent regeneration affected the unfixed, in situ structure of the tectorial membrane. We have recently developed techniques for studying the unfixed tectorial membrane with video-enhanced differential-interference-contrast (DIC) light microscopy. Exposure to a 1500-Hz pure tone at 120 dB SPL for 24 h causes localized damage to the hair cells and tectorial membrane in the mid-proximal region of the basilar papilla. Examination of the unfixed membrane immediately after noise exposure shows that the damage to the tectorial membrane is actually caused by the acoustic trauma and is not an artifact of fixation. After 14 days of recovery, a thick, honeycomb of new matrix has grown from the supporting cells in the basilar papilla and has formed new connections with the stereocilia of surviving and regenerating hair cells. Moreover, this new honeycomb has fused with the remainder of the surrounding, undamaged tectorial membrane, thus reestablishing a continuity in the structure of the membrane across both the damaged and undamaged regions of the basilar papilla.

The effect of acoustic trauma on the tectorial membrane, stereocilia, and hearing sensitivity: possible mechanisms underlying damage, recovery, and protection.

The aim of the present investigation was to determine: 1) the relationship between changes in auditory sensitivity and alterations in stereocilia micromechanics and tectorial membrane morphology after acoustic overstimulation; 2) the rate of growth of a threshold shift in stereocilia following in vitro overstimulation; and 3) if the damaging effects of noise trauma can be reduced by pre-exposure to a low level acoustic stimulus. After exposure to a 1.0 kHz pure tone signal at 105 dB SPL for 72 hours, the threshold of the auditory brainstem response was broadly elevated by approximately 40-50 dB; the inner hair cell stereocilia became less stiff; and morphological alterations were observed in the middle zone of the tectorial membrane. The location of both the stereocilia and tectorial membrane alterations corresponded to the region of the cochlea demonstrating a threshold shift. Following a recovery period from overstimulation, the auditory brainstem response showed some improvement yet a 25 dB threshold shift remained. At this time, swelling of the afferent dendrites beneath the inner hair cells was observed together with scattered outer hair cell loss. Also, the inner hair cell stereocilia regained their normal stiffness characteristics. The in vitro experiments demonstrated that overstimulation reduced the stiffness of the inner and outer hair cell stereocilia bundles. A threshold shift increased systematically with exposure duration and intensity. After 6 minutes of overstimulation, the threshold shift exhibited a plateau whose magnitude was dependent upon the exposure intensity. Stereocilia micromechanics were shown to be dependent on the metabolism of the hair cell. The pre-treatment to a low level acoustic stimulus (81 dB SPL) prior to exposure to a stimulus known to yield a permanent threshold shift resulted in a 20 dB reduction in the threshold shift relative to the group not pre-exposed as well as complete recovery from the threshold shift after 2 months.

How OHC lesions can lead to neural cochlear hypersensitivity.

Several laboratories, including ours, have found that damage to OHCs--but not to IHCs--can produce hypersensitivity of cochlear nerve afferents to low-frequency sound in the presence of hyposensitivity at the characteristic frequency (CF). It is possible to explain the paradox by a mechanical coupling of the OHCs and IHCs through the tectorial membrane, for which there is accumulating experimental evidence. No direct contact between the IHC stereocilia and the tectorial membrane is required.