In the present study, we show that mice doubly heterozygous for Cdh23v-2J
mutations exhibit deafness and abnormal stereocilia in outer and inner hair cells of the organ of Corti by 5 months of age, whereas single heterozygotes lack this pathology. Within the cochlea, Cdh23
are expressed in the inner and outer hair cells (6
). In agreement with their expression pattern, we observed hair-cell specific defects in Cdh23v-2J
double heterozygous mice. Ultrastructural studies have revealed four types of stereocilia links including tip links, top links, shaft or lateral links and ankle links (28
). They are thought to provide cohesiveness to the stereocilia bundle. Ankle links are present in mouse hair-cell stereocilia up to postnatal day 12 and then begin to disappear and are completely absent in adult mice (5
). Cadherin 23 was first reported to be undetectable in stereo-cilia of mice older than postnatal day 30 (29
), raising the possibility that it may be important for the development of stereocilia bundle and may have a role in the formation and maintenance of some transient links, such as ankle links, but not in the long-term maintenance of stereocilia bundle morphology. However, recent studies have indicated that CDH23 was still expressed in mature cochlear hair cells, even when hair cells have lost their kinocilium (30
). It was suggested that its role in adult hair cells might be to form kinocilia links and tip links, which transmit force to mechanically gated ion channels. Protocadherin 15 is expressed in the stereo-cilia of developing mammalian hair cells and persists in adult hair cells along the length of stereocilia, indicating that it might be important for the long-term maintenance of lateral links between stereocilia (5
). The prominent role of cadherins is in mediated cell-cell interactions (14
). Thus, homophilic interaction of CDH23 and PCDH15 proteins might form links that interconnect stereocilia within a bundle. Both proteins could also engage in heterophilic interactions. Previous studies have suggested that interactions among the USH1-proteins myosin VIIa, harmonin, cadherin 23 and SANS may play a fundamental role in differentiation of the actin bundle in stereocilia (29
). A failure of this process leads to stereocilia disorganization, as observed in mouse models (7
), and is thought to be responsible for profound congenital deafness in patients with Usher syndrome (33
). In the present studies, abnormal links between stereocilia could account for the stereocilia defect seen in double heterozygous mice. Loss of Cdh23 and Pcdh15 functions in heterozygous mice is progressive with aging, which indicates a crucial role for these proteins in the long-term maintenance of lateral connections between stereocilia.
Genetic fine mapping suggests that the mouse waltzer mutation (Cdh23v
) is allelic with mdfw
). We previously shown that Cdh23v
might also be allelic to the ahl
). Recent evidence demonstrates that a strain-specific dimorphism of Cdh23
likely underlies the mdfw
). Furthermore, it is known that in mouse, age-related hearing loss is subject to strain-specific effects. We have reported that the C57BL6/J background on which Cdh23v-2J
mutations are carried is susceptible to age-related hearing loss (11
). Mice heterozygous for a presumed null allele of cdh23
) were shown by others to have low-and high-frequency hearing loss at 5–6 weeks of age, the high-frequency component of which worsens with increasing age (30
). However, the hearing loss in these Cdh23v
heterozygotes was dependent upon genetic background. In the present study, we clearly show that the hearing loss in Cdh23v-2J
double heterozygous mice is due to a genetic interaction between Cdh23v-2J
rather than genetic background effects, because all mice tested were on a uniform C57BL/6J background. The observed genetic interaction might be a consequence of functional redundancy, as suggested by the following lines of evidence. First, both Cdh23-
encoded proteins might be involved in lateral connections between stereocilia, because of their potential to participate in homophilic interactivity. Second, the phenotypic abnormalities of double heterozygotes are detected only in structures where both proteins are expressed. Finally, when defective, both proteins produce similar phenotypes.
The cytoplasmic domains of protocadherins, but not classical cadherins, are highly variable and contain various cytoplasmic sequences. It is, therefore, quite reasonable to assume that the protocadherins may have a variety of heterophilic interaction activities. The Cdh23v-2J mutation of the mice examined in the present study truncates 14 extracellular cadherin repeats, the putative transmembrane domain and the cytoplasmic tail. Although the truncated protein could possibly exist in secreted form and function as a dominant negative, the recessive inheritance of Cdh23v-2J argues in favor of a loss of function allele. The mutation in Pcdh15av-3J leads to the truncation of the cytoplasmic domain. As the mutant proteins in Cdh23v-2J and Pcdh15av-3J lack the C-terminal domains, mediation of the assembly via their tail domains is likely compromised. Our data together indicate that the genes encoding cadherin 23 and protocadherin 15 genetically interact to affect the long-term maintenance of the proper organization of the sterocilia bundle. However, we cannot exclude the possibility that they may have some role in their morphogenesis.
Here, we have provided evidence for hearing loss caused by an interaction of cadherin 23 and protocadherin 15 mutations in digenic heterozygotes in both mice and humans. However, the phenotypic outcome of the digenic heterozygotes is different in both species: patients with USH1 are congenitally deaf, whereas mice doubly heterozygous for Cdh23v-2J
have a progressive age-related hearing loss. It has previously been reported that the severity of phenotype in USH1D patients varies widely for the same mutation in the human population, even among members of the same family (2
). This suggests that genetic background may be an important determinant in the extent of the pathology. Different genetic backgrounds could, therefore, be a possible explanation for the difference seen between humans and mice. Elucidation of the causes of this dissimilarity should advance our understanding of the underlying etiology of USH.