**4.1 USH proteins in the inner ear**

The inner ear is composed of the cochlea and vestibular system for hearing and balance, respectively. In the vestibular system, hair cells exist in the maculae of the saccule and utricle and the cristae ampullares of the semicircular canals. In the cochlea, one row of inner hair cells and three rows of outer hair cells exist in the organ of Corti. The inner hair cells are responsible for mechanoelectric transduction, whereas the electromotile outer hair cells also perform an electromechanical transduction, thereby amplifying the sound-evoked vibrations of the entire sensory epithelium (Leibovici et al., 2008). All types of hair cells have stereocilia on their apical surfaces, which are modified microvilli filled with bundles of actin filaments. The stereocilia are well-organized into rows of different lengths and form a staircase-like hair bundle (Figure 4). Along with the hair bundle, there exists a real cilium, called kinocilium, which is filled with microtubules. Various links have been discovered along the entire length of the stereocilia and the kinocilium during development and in

Usher Syndrome: Genes, Proteins, Models, Molecular Mechanisms, and Therapies 305

and PCDH15 at the base of the outer segment by one research group (Ahmed et al., 2003; Reiners et al., 2005a; Maerker et al., 2008). However, other research groups did not find harmonin in the outer segment (Williams et al., 2009), and MYO7A was demonstrated to be predominantly expressed in the RPE cells (Hasson et al., 1995; el-Amraoui et al., 1996; Lopes et al., 2011). USH2 proteins were initially localized to the inner segment, adherens junction, connecting cilium, basal bodies, and synaptic terminus in photoreceptors (Figure 4) (Kremer et al., 2006; Reiners et al., 2006; van Wijk et al., 2006; Maerker et al., 2008; Lagziel et al., 2009). With the antibodies whose specificities have been confirmed in their respective mutant mice, the three USH2 proteins were recently localized to the periciliary membrane complex (PMC) around the connecting cilium (Figure 4) (Liu et al., 2007; Yang et al., 2010; Yang et al., 2011; Zou et al., 2011). Finally, the distribution of clarin-1 in the retina is controversial. One report shows that it is present around the connecting cilium in photoreceptors (Zallocchi et al., 2009), while the other indicates that clarin-1 is restricted to the Muller cells but not

The calycal processes in photoreceptors are thought as an analogous structure to the stereocilia in hair cells (Goodyear and Richardson, 1999). They are well developed in humans, frogs and other species. In mice, only cone photoreceptors have obvious calycal processes (Cohen, 1965; Fetter and Corless, 1987; Rana and Taraszka, 1991). GPR98 and CDH23 are localized at the calycal processes in mouse cone photoreceptors, while whirlin is not evident at this structure in frog photoreceptors (Goodyear and Richardson, 1999; Yang et

The indistinguishable symptoms within the same USH clinical type and the similar symptoms across different USH clinical types indicate that various USH proteins probably participate in the same cellular pathway in a broad sense. Among the USH proteins, harmonin, whirlin and SANS possess multiple protein-protein interaction domains and are proposed to be scaffold proteins in multi-protein complexes. Biochemical assays have indeed revealed the existence of their self-interactions and interactions with most of other USH proteins in vitro (Table 2). Interestingly, the in vitro interactions among different USH1 and/or USH2 proteins exist extensively (Table 2). One USH protein is generally able to interact with at least three other USH proteins. In most cases, different regions of the same protein are involved in its binding to different USH proteins (Table 2). Although these interactions have not been individually confirmed in vivo, harmonin, MYO7A, and CDH23 were recently reported to form a ternary complex in hair cells (Bahloul et al., 2010). Based on these findings, it has been hypothesized that USH proteins form an interacting network, an interactome, in both hair cells and photoreceptors (Richardson et al.; Kremer et al., 2006;

The above hypothesis is supported by the facts that ablation of one USH protein in mice causes mislocation and/or disappearance of at least one other USH protein in hair cells (Table 3). This phenomenon occurs across USH1 and USH2 proteins. Normal distribution of the three USH2 proteins depends on MYO7A and the distribution of some CDH23 isoform at the tip of the stereocilia relies on GPR98 (Table 3). However, the USH1 and USH2 proteins are present at the different interstereociliary links in hair cells during development. Additionally, different USH proteins are localized at two distinct subcellular locations in

photoreceptors (Geller et al., 2009).

**5. The USH protein complexes** 

Reiners et al., 2006; Saihan et al., 2009; Millan et al., 2011).

al., 2010).

adulthood (Goodyear and Richardson, 1999; Goodyear and Richardson, 2003; Goodyear et al., 2005).

The distribution of USH proteins in hair cells vary dramatically from the emergence of stereocilia to their maturation. All USH1 proteins are present either at the tip, the ankle links, the transient lateral links, or the kinociliary links of the stereocilia during the early stage of development. They are then restricted to the tip link and the accessory structures of the tip link, the upper (UTLD) and lower (LTLD) tip link densities, in mature hair cells (Figure 4) (Kussel-Andermann et al., 2000; Senften et al., 2006; Lefevre et al., 2008; Grillet et al., 2009; Bahloul et al., 2010; Caberlotto et al., 2011; Grati and Kachar, 2011). USH2 proteins are localized at the ankle links of the stereocilia (McGee et al., 2006; van Wijk et al., 2006; Michalski et al., 2007; Yang et al., 2010), which is a transient structure existing only during development (Goodyear et al., 2005). Whirlin is also present at the tip of stereocilia in the vestibular and cochlear hair cells all the time (Belyantseva et al., 2005; Delprat et al., 2005; Kikkawa et al., 2005). Clarin-1 was found at the stereocilia on postnatal day 0 (Zallocchi et al., 2009). Besides their location at the stereocilia, some USH proteins were found at the synaptic region of the outer and inner hair cells (Reiners et al., 2005b; van Wijk et al., 2006; Zallocchi et al., 2009), the cell body of the spinal ganglia (Alagramam et al., 2001b; Adato et al., 2002; van Wijk et al., 2006), the supporting cells (Alagramam et al., 2001b; Adato et al., 2005a; Adato et al., 2005b), various nervous fibers (van Wijk et al., 2006), and Reissner's membrane (Wilson et al., 2001; Lagziel et al., 2005). However, these distributions of USH proteins need to be further verified, because the specificity of antibodies used in the studies were not confirmed in their corresponding mutant mice.

#### **4.2 USH proteins in the retina**

In the retina, USH proteins are mainly localized in the photoreceptors (Kremer et al., 2006; Reiners et al., 2006; van Wijk et al., 2006; Liu et al., 2007; Maerker et al., 2008; Yang et al., 2010). The photoreceptor is a highly polarized sensory neuron converting light signals to electrical impulses. It consists of the outer segment, connecting cilium, inner segment, cell body, and synaptic terminus (Figure 4). It contacts Muller cells at the adherens junction (the outer limiting membrane in the retina). Its outer segment is immediately next to the retinal pigment epithelium (RPE) cells.

Compared with the studies in the inner ear, the cellular location of USH proteins is less well defined in the retina. All the USH proteins were once localized in the synaptic ends of photoreceptors (Reiners et al., 2005a; Reiners et al., 2005b; Maerker et al., 2008). However, these results are not conclusive (Williams, 2008; Saihan et al., 2009). They are not supported by the phenotypic analyses in USH mutant mice and the symptom manifestation in USH patients. For instance, ultrastructural abnormalities were not found at the synaptic terminus of photoreceptors in USH mice by electron microscopy (Self et al., 1998; Williams et al., 2009; Yang et al., 2010). No defective ERG waveforms typically resulting from abnormal photoreceptor synaptic transmission have been detected in USH mutant mice (Libby and Steel, 2001; Ball et al., 2003; Libby et al., 2003; Haywood-Watson et al., 2006; McGee et al., 2006; Liu et al., 2007; Yang et al., 2010) or in USH patients.

In addition to the synaptic distribution, MYO7A and SANS were shown to be present around the connecting cilium, harmonin at the outer segment, CDH23 in the inner segment, and PCDH15 at the base of the outer segment by one research group (Ahmed et al., 2003; Reiners et al., 2005a; Maerker et al., 2008). However, other research groups did not find harmonin in the outer segment (Williams et al., 2009), and MYO7A was demonstrated to be predominantly expressed in the RPE cells (Hasson et al., 1995; el-Amraoui et al., 1996; Lopes et al., 2011). USH2 proteins were initially localized to the inner segment, adherens junction, connecting cilium, basal bodies, and synaptic terminus in photoreceptors (Figure 4) (Kremer et al., 2006; Reiners et al., 2006; van Wijk et al., 2006; Maerker et al., 2008; Lagziel et al., 2009). With the antibodies whose specificities have been confirmed in their respective mutant mice, the three USH2 proteins were recently localized to the periciliary membrane complex (PMC) around the connecting cilium (Figure 4) (Liu et al., 2007; Yang et al., 2010; Yang et al., 2011; Zou et al., 2011). Finally, the distribution of clarin-1 in the retina is controversial. One report shows that it is present around the connecting cilium in photoreceptors (Zallocchi et al., 2009), while the other indicates that clarin-1 is restricted to the Muller cells but not photoreceptors (Geller et al., 2009).

The calycal processes in photoreceptors are thought as an analogous structure to the stereocilia in hair cells (Goodyear and Richardson, 1999). They are well developed in humans, frogs and other species. In mice, only cone photoreceptors have obvious calycal processes (Cohen, 1965; Fetter and Corless, 1987; Rana and Taraszka, 1991). GPR98 and CDH23 are localized at the calycal processes in mouse cone photoreceptors, while whirlin is not evident at this structure in frog photoreceptors (Goodyear and Richardson, 1999; Yang et al., 2010).
