**5. The USH protein complexes**

304 Hearing Loss

adulthood (Goodyear and Richardson, 1999; Goodyear and Richardson, 2003; Goodyear et

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

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

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.,

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,

were not confirmed in their corresponding mutant mice.

2006; Liu et al., 2007; Yang et al., 2010) or in USH patients.

**4.2 USH proteins in the retina** 

pigment epithelium (RPE) cells.

al., 2005).

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

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

Usher Syndrome: Genes, Proteins, Models, Molecular Mechanisms, and Therapies 307

cen MYO7A/MyTH4-FERM (Wu et al, 2011.; Adato et al., 2005b) SAM, PBM Harmonin/PDZ1 (Weil et al., 2003; Yan et al., 2010)

PBM Whirlin/PDZ1-PDZ2 (Adato et al., 2005a; van Wijk et al.,

PBM Whirlin/PDZ1-PDZ2 (Adato et al., 2005a; van Wijk et al.,

PDZ1-PDZ2 USH2A/PBM (Adato et al., 2005a; van Wijk et al.,

PDZ1-PDZ2 GPR98/PBM (Adato et al., 2005a; van Wijk et al.,

In hair cells, the normal cellular localization of harmonin requires the presence of all other USH1 proteins, and loss of harmonin seems not to affect the localization of other USH1 proteins (Table 3), indicating that harmonin is dispensable for locating these USH1 proteins to their normal position in cells. In contrast, CDH23 is relatively independent on other USH1 proteins, and its loss results in mislocalization of the two putative scaffold proteins, harmonin and SANS (Table 3). Therefore, CDH23 may play a crucial role in anchoring/tethering USH1

Besides the known USH proteins, many other putative components in the USH complexes has been identified. These components are able to interact with at least one of the USH proteins as shown by biochemical assays. For the currently known USH2-interacting proteins, please see the review (Yang et al., 2011). However, additional experiments are necessary to verify the existence of these putative components in the USH complexes in vivo

PDZ3 Whirlin/PDZ1-PDZ2, PR-PDZ3 (Delprat et al., 2005; Yang et al.,

proteins. Harmonin and SANS may help hold the USH1 proteins in the complex.

2006; Yang et al., 2010)

2006; Yang et al., 2010)

2006; Yang et al., 2010)

2006; Yang et al., 2010)

2010)

**Proteins/domains Interacting proteins/domains References** 

Not determined CDH23/cytoplasmic region (Caberlotto et al., 2011) Not determined PCDH15/CD2, CD3 (Caberlotto et al., 2011) PBM Whirlin/PDZ1-PDZ2 (Maerker et al., 2008) cen SANS/cen (Adato et al., 2005b)

region MYO7A/MyTH4-FERM (Michalski et al., 2007) PBM Harmonin/PDZ1 (Reiners et al., 2005b)

region MYO7A/MyTH4-FERM (Michalski et al., 2007) PBM Harmonin/PDZ1 (Reiners et al., 2005b)

Not determined MYO7A/not determined (Delprat et al., 2005) PDZ1-PDZ2 SANS/PBM (Maerker et al., 2008)

**SANS** 

**USH2A**  Cytoplasmic

**GPR98**  Cytoplasmic

**Whirlin** 

PDZ1-PDZ2, PR-

Table 2. Interactions among USH proteins

and reveal their relationship with USH.

photoreceptors, the PMC and the synapse. Due to these different cellular locations of USH proteins, it is reasonable to propose that more than one USH protein complex exist and they play different but highly related roles in a broad cellular process (Williams, 2008; Yang et al., 2011).


photoreceptors, the PMC and the synapse. Due to these different cellular locations of USH proteins, it is reasonable to propose that more than one USH protein complex exist and they play different but highly related roles in a broad cellular process (Williams, 2008; Yang et al.,

MyTH4-FERM SANS/cen (Wu et al., 2011; Adato et al., 2005b)

PDZ2 PCDH15/CD1 PBM (Adato et al., 2005b; Reiners et al.,

PDZ1/2, CC2 Harmonin/PBM, CC2 (Siemens et al., 2002; Adato et al.,

PDZ1/3 SANS/SAM, PBM (Adato et al., 2005b; Yan et al.,

PDZ1 USH2A/PBM (Reiners et al., 2005b) PDZ1 GPR98/PBM (Reiners et al., 2005b)

not determined MYO7A/tail (Bahloul et al., 2010)

EC1-3 PCDH15/EC1 (Kazmierczak et al., 2007)

ECs CDH23/ECs (Siemens et al., 2004; Kazmierczak

CD1 PBM Harmonin/PDZ2 (Adato et al., 2005b; Reiners et al.,

region SANS/not determined (Caberlotto et al., 2011)

region MYO7A/SH2 (Senften et al., 2006)

EC1 CDH23/EC1-3 (Kazmierczak et al., 2007) CD2/CD3 SANS/not determined (Caberlotto et al., 2011) ECs PCDH15/ECs (Kazmierczak et al., 2007)

2 PBMs Harmonin/N-terminus, PDZ1, PDZ2

(Boeda et al., 2002; Siemens et al., 2002; Grillet et al., 2009; Pan et al.,

(Boeda et al., 2002; Siemens et al., 2002; Grillet et al., 2009; Pan et al.,

2009; Bahloul et al., 2010)

2005b; Senften et al., 2006)

2009; Bahloul et al., 2010)

2005b; Senften et al., 2006)

2010)

2005b)

et al., 2007)

**Proteins/domains Interacting proteins/domains References** 

MyTH4-FERM Harmonin/PDZ1 (Boeda et al., 2002) Tail CDH23/not determined (Bahloul et al., 2010) SH2 PCDH15 (Senften et al., 2006)

MyTH4-FERM USH2A/cytoplasmic region (Michalski et al., 2007) MyTH4-FERM GPR98/cytoplasmic region (Michalski et al., 2007) Not determined Whirlin/not determined (Delprat et al., 2005)

PDZ1 MYO7A/MyTH4-FERM (Boeda et al., 2002)

2011).

**MYO7A** 

**Harmonin** 

N-terminus,

**CDH23** 

Cytoplasmic

**PCDH15**  Cytoplasmic

PDZ1/2 CDH23/PBMs


Table 2. Interactions among USH proteins

In hair cells, the normal cellular localization of harmonin requires the presence of all other USH1 proteins, and loss of harmonin seems not to affect the localization of other USH1 proteins (Table 3), indicating that harmonin is dispensable for locating these USH1 proteins to their normal position in cells. In contrast, CDH23 is relatively independent on other USH1 proteins, and its loss results in mislocalization of the two putative scaffold proteins, harmonin and SANS (Table 3). Therefore, CDH23 may play a crucial role in anchoring/tethering USH1 proteins. Harmonin and SANS may help hold the USH1 proteins in the complex.

Besides the known USH proteins, many other putative components in the USH complexes has been identified. These components are able to interact with at least one of the USH proteins as shown by biochemical assays. For the currently known USH2-interacting proteins, please see the review (Yang et al., 2011). However, additional experiments are necessary to verify the existence of these putative components in the USH complexes in vivo and reveal their relationship with USH.


+: existence of mislocalization, -: normal localization, +/-, contradictory results

Table 3. Interdependence of USH proteins in hair cells

Usher Syndrome: Genes, Proteins, Models, Molecular Mechanisms, and Therapies 309

The severe and early-onset hearing phenotypes in various USH1 and USH2 mouse models make it relatively easier to decipher the functions of USH complexes in the inner ear than in the retina. The following will focus on the three main cellular processes generally believed to involve the USH complexes. Disruption of these USH functions is thought to be the

During development, at the apex of hair cells, microvilli grow into stereocilia by recruiting more actin filaments. These stereocilia are bundled with transient lateral links and are connected with the kinocilium through kinociliary links. Following the establishment of the planar cell polarity, the kinocilium moves from the center to the periphery of the cell, and the stereocilia elongate differentially. The staircase-shape hair bundle is eventually formed. At the same time, the transient lateral links are gradually substituted by two distinct sets of interstereociliary links. They are the horizontal top connectors and the ankle links, close to the tip and base of the hair bundle, respectively (Figure 4). The tip links emerge, which are fibrous connections between the tip of medium and low stereocilia and the side of the neighboring taller stereocilia (Figure 4). Finally, the stereocilia grow both in length and in width and reach their mature size. In rodent mature cochlear hair cells, the ankle links and the kinociliary links disappear with the regression of the kinocilium (Frolenkov et al., 2004;

CDH23 (Siemens et al., 2004; Lagziel et al., 2005; Michel et al., 2005; Rzadzinska et al., 2005; Lefevre et al., 2008) and PCDH15 (Goodyear et al., 2010; Webb et al., 2011; Lefevre et al., 2008) are localized at the transient lateral links and kinociliary links during early development of hair cells. In their mutant mice, hair bundles are usually splayed into several clumps; kinocilium is mispositioned and disconnected with the hair bundle (Lefevre et al., 2008), indicating that CDH23 and PCDH15, as components of the interstereociliary links, are important for hair bundle cohesion and that loss of the connection between the stereocilia and kinocilium causes the misorientation of the hair bundle. Interestingly, the mutant mouse models of all five USH1 genes share such similar phenotypes. This could be explained by the idea that the five USH1 proteins coordinate in this function. The PST domain of harmonin b binds to and bundles actin filaments (Boeda et al., 2002). MYO7A is a high duty ratio motor, which binds to actin filament strongly. Therefore, these two actinbinding proteins may anchor their interacting partners, CDH23 and PCDH15, to the actin bundle in the stereocilia of hair cells (Table 2). In *Ush1g-/-* mice, cohesion of stereocilia is disrupted. In *Ush1gfl/flMyo7a-cre+/-* mice, whose expression of SANS is disturbed only after birth, the stereocilia stay cohesive (Caberlotto et al., 2011). Therefore, SANS plays a role in stereocilia cohesion during the prenatal period. It may be involved in the organization of

All three USH2 proteins, USH2A, GPR98, and whirlin, are positioned at the ankle links of hair cells. Among these proteins, USH2A and GPR98 probably interact with each other or with some unidentified cell adhesion proteins to form the ankle links. Whirlin interacts with USH2A and GPR98 through the PDZ domain-mediated binding to anchor them at the base of the stereocilia. In the absence of GPR98, the ankle links are missing. Thus far, the

other USH1 proteins through directly interacting with them (Table 2).

**6. Functions of the USH complexes** 

molecular mechanisms underlying USH.

Goodyear et al., 2005; Nayak et al., 2007).

**6.1 Hair bundle cohesion** 
