*4.2. TRIOBP-5 in the Inner Ear and Deafness*

While the majority of *TRIOBP* mutations found in patients with profound hearing loss lie within the reading frame of *TRIOBP-4* (Table 1), these would also affect the TRIOBP-5 and 6 proteins. Additionally, patients with moderate and/or progressive hearing loss have been described that possess both a mutation in *TRIOBP-4* and a p.G1672\* mutation on the other *TRIOBP* allele, which would affect only the longer splice variants [59,61]. Consistent with this, while mice who lack both TRIOBP-4 and 5 show profound deafness [50], those engineered to express TRIOBP-4, but not 5, instead display a progressive form of deafness [18]. Together, these findings strongly imply that while TRIOBP-4 is essential for prelingual hearing ability, specific loss of the longer splice variants is also required for maintenance of hearing.

TRIOBP-5 is expressed in the same inner ear cell types as TRIOBP-4, with both being found in the stereocilia rootlets [50]. In contrast to TRIOBP-4, however, TRIOBP-5 is predominantly found in the lower parts of the rootlet, below the apical surface [18]. The specific role of TRIOBP-5 in the ear has been studied using various *TRIOBP*-deficient mice. While deletion of both *TRIOBP-1* and *TRIOBP-5* is lethal [50], mice lacking two *TRIOBP-5* specific exons are viable, as are heterozygous mice that can express TRIOBP-1 from one allele and TRIOBP-4 from the other [18]. These *TRIOBP-5*-deficient mice still express TRIOBP-4 in the stereocilia and retain residual hearing for at least 4–8 weeks [18]. This contrasts with the profound deafness of *TRIOBP-4*-deficient mice [50], indicating a unique role of TRIOBP-5, which is also essential for hearing. Detailed analysis of the *TRIOBP-5* knockout mice revealed that stereocilia appear to form normally, but then become increasingly disorganized over time. Specifically, some fuse together or are missing, while others appear thin and fragmented compared to those of wild-type animals [18]. The stereocilia are also seen to be less stiff, and to rotate less freely than wild-type ones [18]. Therefore, while TRIOBP-4 appears to be required to form stereocilia rootlets and elongate them into tight actin bundles (a role indispensable for hearing), TRIOBP-5 instead plays a separate, later role in widening and giving structure to the stereocilia (loss of which leads to progressive hearing loss) [18,50].

Interestingly, this role of TRIOBP-5 in modeling of the rootlets is retained in mice that express incomplete TRIOBP-5 (terminating after the PH domain), however, they do not gain the usual resilience [18]. Such mice may therefore reflect patients with mutations like p.G1672\*, who have moderate progressive hearing loss, but not the profound hearing loss associated with mutations in *TRIOBP-4* [18,59,61]. This also implies that the role of TRIOBP-5 in the stereocilia is likely to involve its coiled-coil domains. One possible explanation for this is that these domains interact with Pejkavin, a protein also required for bundling of actin in the inner ear and for hearing, which was seen to interact with this region of TRIOBP-1 [20,65].

It is likely that TRIOBP-6 could also be involved in this process, but this remains untested due to lack of a known murine TRIOBP-6 species.
