**6.3 Protein and organelle transport**

310 Hearing Loss

dependence of the ankle links on USH2A and whirlin has not been examined. In the wildtype mouse, the stereocilia of outer hair cells are organized into a V-shaped staircase-like hair bundle. However, in all three *Ush2* mutant mice, the outer hair cells show various disorganized stereocilia and abnormal U-shape hair bundles (Mburu et al., 2003; McGee et al., 2006; Liu et al., 2007; Michalski et al., 2007; Yang et al., 2010). Accordingly, as components of the ankle links, the three USH2 proteins probably contribute to hair bundle

The stereocilia of hair cells are the cellular organelle conducting mechanotransduction. The vibration of the basilar membrane and tectorial membrane or the motion of endolymphatic fluid induces the hair bundle deflection. When the deflection is toward the longest stereocilia (the positive or excitatory direction), the transduction channels are open. The influx of Ca2+ and K+ through the channels elicits changes of the membrane potential and glutamate release at the ribbon synapse in hair cells. When the hair bundle moves away from the longest stereocilia (the negative or inhibitory direction), the transduction channels close, and the membrane potential and transmitter release resume their resting statuses. Although the molecular machinery of mechanotransduction is not well understood, the 'gating spring' model is popular in the field. In this model, the tip link, whose axis is parallel to the direction of the mechanical sensitivity of the hair bundle, is thought as a sensor to the stretch of the hair bundle. Alternatively, an unknown structure attached to the tip link fulfills this function (Vollrath et al., 2007; Gillespie and Muller, 2009). The transduction channel was recently localized to the plasma membrane at the lower end of the tip link in

In mature hair cells, CDH23 (Siemens et al., 2004; Sollner et al., 2004) and PCDH15 (Ahmed et al., 2006) were found associated with the tip links. CDH23 is mainly at the upper part and PCDH15 at the lower part of the links (Kazmierczak et al., 2007; Alagramam et al., 2011). In *Cdh23V-2J* and *Pcdh15aw-6J* mice, the tip links are missing. Additionally, the response of the mechanotransduction is reduced. In the absence of stimulus, a fraction of transduction channels keep open in the wild-type hair cells, due to the resting tension of the tip links. However, the transduction channels in these two mutants do not open or take up the styryl dye FM1-43 at rest (Senften et al., 2006; Alagramam et al., 2011). Therefore, CDH23 and PCDH15 are believed to be components of the tip links and to participate in

At the two ends of the tip link immediately beneath the stereocilia plasma membrane, there are electron-dense complexes, the UTLD and LTLD (Figure 4). Harmonin and MYO7A are present at the UTLD (Grillet et al., 2009; Michalski et al., 2009; Caberlotto et al., 2011; Grati and Kachar, 2011). In *Myo7a6J, Myo7a4626SB*, *Ush1cdfcr*, and *Ush1cdfcr-2J* mice, the adaptation of mechanotransduction, a process for the hair cells to recover their sensitivity under sustained mechanical stimulation, was found consistently abnormal, while the amplitude of mechanotransduction responses is sometimes normal (Kros et al., 2002; Grillet et al., 2009; Michalski et al., 2009). These results suggest that harmonin and MYO7A are involved in the transduction adaptation. SANS may exist at both the LTLD and UTLD (Caberlotto et al., 2011; Grati and Kachar, 2011). Its loss in hair cells (*Ush1g-/-*) causes elimination of the tip links and reduction in both the amplitude and sensitivity of the transduction currents

cohesion as well.

**6.2 Mechanotransduction** 

the stereocilia (Beurg et al., 2008).

mechanotransduction in mature hair cells.

In photoreceptors, the outer segment is a large specialized cilium filled with many flat membrane disks, where phototransduction occurs (Figure 4). This cellular compartment undergoes continuous and rapid renewal (Young, 1967; LaVail, 1976; Young, 1976; Besharse and Hollyfield, 1979), which requires a large amount of proteins and membrane lipids to be synthesized in the inner segment and to be quickly transported to the base of the outer segment through the connecting cilium (Figure 4). The removal of the old outer segment is achieved through phagocytosis by RPE cells. In addition, in both photoreceptors and RPE cells, several proteins, involved in phototransduction and retinoid cycle, translocate between two different cellular compartments in response to light (Artemyev, 2008; Slepak and Hurley, 2008; Lopes et al., 2011).

Among USH proteins, MYO7A is an actin-based motor. In the retina, it is expressed in both RPE cells and photoreceptors. In RPE cells, MYO7A is essential for the transport of phagosomes to their degradation apparatus (Gibbs et al., 2003), tethering melanosomes during their movement (Gibbs et al., 2004), and the translocation of RPE65 responding to light exposure (Lopes et al., 2011). In photoreceptors, MYO7A is present along the connecting cilium. Loss of MYO7A was found to delay the transport of opsin from the inner to the outer segment (Liu et al., 1999) and the transducin translocation from the outer to the inner segment after light exposure (Peng et al., 2011). In hair cells, without MYO7A, all USH2 proteins are mislocalized from the ankle links (Table 3), suggesting that MYO7A may transport the USH2 proteins. These lines of evidence establish the notion that MYO7A may function in protein and organelle transport in various cells in the retina and the inner ear.

USH2 proteins are positioned at the PMC in mammalian photoreceptors, which is an analogous structure to the periciliary ridge complex (PRC) in frogs (Peters et al., 1983). The PRC is a morphologically-specialized structure with a symmetrical array of 9 ridges and 9 grooves. It has been proposed, based on immunocytochemistry and freeze-fracture electron microscopy, as the membrane fusion site for post-Golgi vesicles carrying opsin and docosahexaenoyl (DHA)-phospholipids before these cargos are transported from the inner to the outer segment (Peters et al., 1983; Papermaster et al., 1986; Rodriguez de Turco et al., 1997; Papermaster, 2002). Additionally, Rab8, rac1, Sec8, moesin, syntaxin 3 and SNAP-25 have been localized around the PRC in frog photoreceptors (Deretic et al., 2004; Mazelova et al., 2009). These proteins are proposed, though not verified using mouse genetics, to

Usher Syndrome: Genes, Proteins, Models, Molecular Mechanisms, and Therapies 313

these drugs and the low efficiency of their read-through activities set a hindrance for their further application to patients. A recent report has shown that PTC124, a drug unrelated to aminoglycosides, has a relatively low cellular toxicity and high read-through efficacy

The nutritional supplementation, daily intakes of vitamin A at a dose of 15,000 international units (IU) and vitamin E less than 400 IU, is thought to be a potential effective therapy for retinitis pigmentosa (Berson et al., 1993; Berson, 2000). Although it has already been applied to patients, this vitamin A supplement therapy is still under debate and its underlying

The research on USH has made tremendous progress since the discovery of its first causative gene, *MYO7A*, in 1995. Currently, nine genes have been identified responsible for this genetic disease. From the functional domain analysis, these genes have been proposed to participate in trafficking, scaffolding, cell adhesion, and signaling in cells. Many spontaneous and transgenic mouse, rat, and zebrafish models are available now. The majority of these animal models reproduce the hearing and balance problems in USH patients. However, not many of them manifest retinal degeneration, which is one of the typical symptoms in USH patients. The reason for this discrepancy is not clear. But lack of retinal phenotypes in these animal models hinders our studies on retinitis pigmentosa in USH patients. A large body of evidence from biochemical and cellular localization studies demonstrate that USH proteins are organized into multi-component complexes mainly in hair cells and photoreceptors. They play a role in hair bundle cohesion, mechanotransduction, and, possibly, protein/organelle transport in vivo. USH is an incurable disease. Effective treatments using different approaches are still being sought and

Aarnisalo AA, Pietola L, Joensuu J, Isosomppi J, Aarnisalo P, Dinculescu A, Lewin AS,

Adato A, Michel V, Kikkawa Y, Reiners J, Alagramam KN, Weil D, Yonekawa H, Wolfrum

Adato A, Vreugde S, Joensuu T, Avidan N, Hamalainen R, Belenkiy O, Olender T, Bonne-

ribozyme induced apoptosis in the mouse cochlea. Hear Res 230:9-16. Adato A, Lefevre G, Delprat B, Michel V, Michalski N, Chardenoux S, Weil D, El-Amraoui

Flannery J, Hauswirth WW, Sankila EM, Jero J (2007) Anti-clarin-1 AAV-delivered

A, Petit C (2005a) Usherin, the defective protein in Usher syndrome type IIA, is likely to be a component of interstereocilia ankle links in the inner ear sensory cells.

U, El-Amraoui A, Petit C (2005b) Interactions in the network of Usher syndrome

Tamir B, Ben-Asher E, Espinos C, Millan JM, Lehesjoki AE, Flannery JG, Avraham KB, Pietrokovski S, Sankila EM, Beckmann JS, Lancet D (2002) USH3A transcripts encode clarin-1, a four-transmembrane-domain protein with a possible role in

(Goldmann et al., 2011).

mechanism is unknown.

explored.

**9. References** 

Hum Mol Genet 14:3921-3932.

type 1 proteins. Hum Mol Genet 14:347-356.

sensory synapses. Eur J Hum Genet 10:339-350.

**8. Summary and perspective** 

participate in and/or regulate the docking and membrane fusion of post-Golgi vesicles to the plasma membrane at the PRC. Therefore, the USH2 complex at the PMC might play either a direct or indirect role in the docking between the post-Golgi vesicles and plasma membrane at the base of the connecting cilium (Roepman and Wolfrum, 2007; Maerker et al., 2008). This proposed function can also be applied in hair cells. The ankle-links exist when stereocilia grow and differentiate from small microvilli. At this time, many vesicles are at the base of stereocilia (Forge et al., 1997; Hasson et al., 1997), which could be the post-Golgi vesicles carrying proteins and membrane lipids from the cell body to the growing stereocilia. Supportively, the *Gpr98* knockout mouse shows delocalization of some CDH23 long isoforms at the tip of the stereocilia and, possibly, loss of some apical links between the stereocilia (Michalski et al., 2007). However, solid evidence supporting this putative function of the USH2 complex is still scarce. For instance, obvious mislocalization of rhodopsin has not been observed in whirlin knockout and *Ush2a* knockout mice (Liu et al., 2007; Yang et al., 2010), and vesicles fused with the plasma membrane have not been demonstrated at the ankle links.

### **7. Therapeutic studies**

Because of the widespread clinical application of the well-developed cochlear implant for hearing loss (Pennings et al., 2006; Liu et al., 2008), more attention is focused on seeking effective treatments for retinitis pigmentosa in USH. Next, I will address the current progress in studies on gene therapy, drug application, cell transplantation, and nutritional supplements (Yang et al., 2011).

Human neural progenitor cells from the post mortem fetal cortical brain have been tested in the *Ush2*a knockout mouse (Lu et al., 2009). The progenitor cells were transplanted between photoreceptors and RPE cells. There, they delayed the cellular changes in photoreceptors and alleviated retinal functional deterioration. However, due to the short follow-up time after the treatment, the study did not examine whether the treatment can rescue photoreceptor loss in this animal model.

Compared to the cell-based therapy, replacement of the mutant gene in the retina is straightforward. The efficiency and efficacy of a lentivirus-mediated gene replacement of MYO7A have been studied in the *Myo7a4626SB* mouse (Hashimoto et al., 2007). Although the delivery of MYO7A into photoreceptors and RPE cells is not quite efficient, the treated mutant retina does show correction of the histological phenotypes in these two cells. In addition, our laboratory utilized a combination of AAV and a photoreceptor-specific promoter to efficiently target the USH2D gene, whirlin, into both rod and cone photoreceptors. The transgenic whirlin was found to restore the changes of USH2A and GPR98 expression in the whirlin knockout retina (Zou et al., 2011). These encouraging progresses in the USH1B and USH2D mouse models lay a solid foundation for a further and detailed exploration of gene therapy for these and other USH subtypes.

Aminoglycosides and their derivatives can induce a read-through of nonsense mutations by inserting an amino acid at the stop codon. These drugs have been tested in vitro, in cell cultures and in retinal explants to suppress the nonsense mutations found in USH1F (PCDH15) and USH1C (harmonin) patients (Rebibo-Sabbah et al., 2007; Nudelman et al., 2009; Goldmann et al., 2010; Nudelman et al., 2010). However, the high cellular toxicity of these drugs and the low efficiency of their read-through activities set a hindrance for their further application to patients. A recent report has shown that PTC124, a drug unrelated to aminoglycosides, has a relatively low cellular toxicity and high read-through efficacy (Goldmann et al., 2011).

The nutritional supplementation, daily intakes of vitamin A at a dose of 15,000 international units (IU) and vitamin E less than 400 IU, is thought to be a potential effective therapy for retinitis pigmentosa (Berson et al., 1993; Berson, 2000). Although it has already been applied to patients, this vitamin A supplement therapy is still under debate and its underlying mechanism is unknown.
