Characterization of the Usher Syndrome gene CDH23: implications for mechanosensation in the vertebrate inner ear

Deafness is the most common form of sensory impairment afflicting the human population. Approximately one in eight hundred children is born with serious hearing impairment and more than half of these cases are likely due to single gene defects. In addition to hearing loss, mutations in some genes cause Usher Syndrome, not only affecting the auditory apparatus but also causing visual impairment eventually leading to blindness. Several genetic loci have been linked to Usher Syndrome Type I, the most severe form of the disease, and so far five of the relevant genes have been identified. Understanding their molecular role in the context of ear and retina physiology will be invaluable to the design of effective therapies against this devastating disease. Some forms of Usher Syndrome as well as other hearing disorders are caused by defects in the inner ear that contains the end organs for the perception of sound waves, the cochlea, and for the detection of gravity and acceleration, the vestibule. Both end organs contain mechanosensory hair cells that are named after actin rich stereocilia projecting from their apical surface. The stereocilia contain mechanically gated ion channels that open or close upon deflection of the stereocilia. This in turn triggers ion influx into the hair cells, causing changes in cell polarization and alterations in the rate of neurotransmitter release from the hair cells onto innervating neurons. The mechanically gated transduction channel implicated in this event has remained elusive. Mechanical gating of the transduction channel is believed to be triggered by thin filaments, the tip links, connecting adjacent stereocilia into a bundle. It has been suggested that these connector molecules are being stretched during hair cell stimulation, thereby actively pulling open the transduction channel. Although these filaments are clearly detectable on the ultrastructural level, their molecular nature has remained elusive. One molecule that might participate in mechanoelectrical transduction is the transmembrane protein cadherin 23 (CDH23). Mutations in its gene can cause Usher Syndrome, non-syndromic forms deafness and age-related hearing loss in human patients. Mice and zebrafish that carry mutations in the orthologous genes show splayed stereocilia bundle morphology, arguing for a function of the protein product in the cell compartment harboring the transduction channel. Furthermore, CDH23 is large enough to be the tip link, the extracellular filament proposed to gate the mechanotransduction channel.
Here we show that antibodies against CDH23 label the entire stereocilia bundle during hair cell morphogenesis. In mature hair cells CDH23 labelling is confined to the tip links. Further, CDH23 has biochemical properties similar to those of the tip link. In cell-aggregation experiments CDH23 displays Ca2+-dependent, homophilic adhesion potential, an attribute typically observed for members of the cadherin superfamily, which may explain how adjacent stereocilia are linked together. Moreover, CDH23 forms a complex with myosin 1c (MYO1C), the only known component of the mechanotransduction apparatus, suggesting that CDH23 and MYO1C cooperate to regulate the activity of mechanically gated ion channels in hair cells.
Computer assisted alignments with sequences encoding the cytosolic domain of CDH23 reveal two putative PDZ-binding motifs. Others and we can show that CDH23 interacts with the product of a second Usher Syndrome gene, harmonin. Two PDZ domains within harmonin interact with two complementary binding surfaces in the CDH23 cytoplasmic domain. One of the binding surfaces is disrupted by sequences encoded by an alternatively spliced CDH23 exon that is expressed in hair cells, but not in any other tissue analyzed so far. In the ear, harmonin is expressed in the stereocilia of developing hair cells. Since mice with a targeted deletion of the harmonin gene have been reported to phenocopy the splayed stereocilia bundle morphology observed in CDH23 deficient mice, the complex of the two Usher Syndrome proteins is predicted to be important for the stereocilia bundle. Whether the harmonin–CDH23 complex might be involved in mechanotransduction is unclear, since harmonin´s presence in mature stereocilia has not been reported yet.
We concluded that CDH23 may serve a dual function in auditory hair cells: together with harmonin the molecule is important to shape the hair bundle during hair bundle morphogenesis and in mature stereocilia the molecule is part of the tip link complex.