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.The sensory epithelium of the cochlea, the organ of Corti (Fig. 1), contains two types of hair cell, the purely sensory inner hair cells (IHCs) and the electromotile, sensorimotor outer hair cells (OHCs). These cells are critically positioned between two strips of ECM, the basilar membrane (BM) and the tectorial membrane (TM). Signal processing in the cochlea is initiated when sound-induced changes in fluid pressure displace the BM in the transverse direction, causing radial shearing displacements between the surface of the organ of Corti (the reticular lamina) and the overlying TM (1). The radial shear is detected by the hair bundles of the IHCs and the OHCs (2), with the stereocilia of the OHC hair bundles forming an elastic link between the organ of Corti and the overlying TM (3). Deflection of the stereocilia gates the hair cell’s mechanoelectrical transducer (MET) channels, thereby initiating a MET current (4) that promotes active mechanical force production by the OHCs, which, in turn, influences mechanical interactions between the TM and the BM (5, 6). This nonlinear frequency-dependent enhancement process, which boosts the sensitivity of cochlear responses to low-level sounds and compresses them at high levels, is known as the cochlear amplifier (7).Open in a separate windowFig. 1.Schematic cross-section of WT cochlea. Spiral lamina (SLAM), spiral ligament (SLIG), inner pillar cells (IPC), outer pillar cells (OPC), Deiters'' cells (DC), phalangeal process of DC (PhP), Claudius cells (CC), OHC, IHC, reticular laminar (RL), spiral limbus (SL), and major noncellular elements (BM and TM).Whereas the hair bundles of the OHCs are imbedded into the TM and therefore directly excited by relative displacement of the undersurface of the TM and the reticular lamina, those of the IHCs are not in direct contact with the TM, and the way in which they are driven by motion of the BM remains unclear. Intracellular recordings of the receptor potentials in IHCs indicate that the bundles are velocity-coupled (to fluid flow) at low frequencies and displacement-coupled at higher frequencies of stimulation (2, 8, 9). Direct measurements of the motion of the reticular lamina and the lower surface of the TM in an ex vivo preparation of the guinea pig cochlea provide evidence that, at frequencies below 3 kHz, counterphase transverse movements of the two surfaces generate pulsatile fluid movements in the subtectorial space that could drive the hair bundles of the IHCs (10). At higher frequencies, the two surfaces move in phase, and radial shear alone is thought to dominate. Theoretical studies (11) reveal that the boundary layers will be vanishingly thin at high frequencies, that the fluid in the gap between the TM and the reticular lamina will be inviscid, and that the hydrodynamic forces on the hair bundle will be inertial. Although an overlying TM that is not directly attached to a hair bundle does not apply torque to the hair bundle (11), the inertial force of the fluid driving the hair bundle depends on its mass and therefore the size of the gap between the reticular lamina and the TM (11, 12).The TM is composed of radially arrayed collagen fibrils that are imbedded in a noncollagenous matrix composed of a number of different glycoproteins, including Tecta, Tectb, otogelin, otolin, and Ceacam16 (13–16). Mutations in Tecta cause recessive (DFNB21) and dominant (DFNA8/12) forms of human hereditary deafness (17–19), and a dominant missense mutation in Ceacam16 (DFNA4) has been identified recently as a cause of late-onset progressive hearing loss in an American family (15). Mutations in Tecta are one of the most common causes of autosomal-dominant, nonsyndromic hereditary hearing loss (20), and mouse models for the recessive (21) and dominant (22) forms of deafness arising from mutations in Tecta have been created. Together with data from a Tectb-null mutant mouse (23), these studies have provided evidence that the TM plays multiple roles in hearing (24). Although much is known about the structure of the TM, an ECM that is unique to the cochlea, relatively little is known about how it attaches to the apical surface of the cochlear epithelium. Otoancorin, a product of the DFNB22 locus, is expressed on the apical surface of the spiral limbus and has been suggested to mediate TM attachment to this region of the cochlear epithelium (25). In this study, we use gene targeting to inactivate otoancorin. This provides a mouse model for DFNB22, reveals a loss of IHC sensitivity as the primary cause of deafness, and isolates a specific role for the limbal attachment of the TM in driving the hair bundles of the IHCs.