TRIOBP-5 sculpts stereocilia rootlets and stiffens supporting cells enabling hearing.

TRIOBP remodels the cytoskeleton by forming unusually dense F-actin bundles and is implicated in human cancer, schizophrenia, and deafness. Mutations ablating human and mouse TRIOBP-4 and TRIOBP-5 isoforms are associated with profound deafness, as inner ear mechanosensory hair cells degenerate after stereocilia rootlets fail to develop. However, the mechanisms regulating formation of stereocilia rootlets by each TRIOBP isoform remain unknown. Using 3 new Triobp mouse models, we report that TRIOBP-5 is essential for thickening bundles of F-actin in rootlets, establishing their mature dimensions and for stiffening supporting cells of the auditory sensory epithelium. The coiled-coil domains of this isoform are required for reinforcement and maintenance of stereocilia rootlets. A loss of TRIOBP-5 in mouse results in dysmorphic rootlets that are abnormally thin in the cuticular plate but have increased widths and lengths within stereocilia cores, and causes progressive deafness recapitulating the human phenotype. Our study extends the current understanding of TRIOBP isoform-specific functions necessary for life-long hearing, with implications for insight into other TRIOBPopathies.

Mechanical stress-induced reactive gliosis in the auditory nerve and cochlear nucleus.

OBJECT: Hearing levels following microsurgical treatment gradually deteriorate in a number of patients treated for vestibular schwannoma (VS), especially in the subacute postoperative stage. The cause of this late-onset deterioration of hearing is not completely understood. The aim of this study was to investigate the possibility that reactive gliosis is a contributory factor. METHODS: Mechanical damage to nerve tissue is a feature of complex surgical procedures. To explore this aspect of VS treatment, the authors compressed rat auditory nerves with 2 different degrees of injury while monitoring the compound action potentials of the auditory nerve and the auditory brainstem responses. In this experimental model, the axons of the auditory nerve were quantitatively and highly selectively damaged in the cerebellopontine angle without permanent compromise of the blood supply to the cochlea. The temporal bones were processed for immunohistochemical analysis at 1 week and at 8 weeks after compression. RESULTS: Reactive gliosis was induced not only in the auditory nerve but also in the cochlear nucleus following mechanical trauma in which the general shape of the auditory brainstem response was maintained. There was a substantial outgrowth of astrocytic processes from the transitional zone into the peripheral portion of the auditory nerve, leading to an invasion of dense gliotic tissue in the auditory nerve. The elongated astrocytic processes ran in parallel with the residual auditory neurons and entered much further into the cochlea. Confocal images disclosed fragments of neurons scattered in the gliotic tissue. In the cochlear nucleus, hypertrophic astrocytic processes were abundant around the soma of the neurons. The transverse diameter of the auditory nerve at and proximal to the compression site was considerably reduced, indicating atrophy, especially in rats in which the auditory nerve was profoundly compressed. CONCLUSIONS: The authors found for the first time that mechanical stress to the auditory nerve causes substantial reactive gliosis in both the peripheral and central auditory pathways within 1-8 weeks. Progressive reactive gliosis following surgical stress may cause dysfunction in the auditory pathways and may be a primary cause of progressive hearing loss following microsurgical treatment for VS.

Silencing p27 reverses post-mitotic state of supporting cells in neonatal mouse cochleae.

The post-natal cochlear mammalian epithelium have no capacity to proliferate in tissue, however, dissociated supporting cells exhibit the ability to divide and trans-differentiate into new hair cells in vitro, with this process found to be correlated with the downregulation of the cyclin-dependent kinase inhibitor p27(kip1). Here we show that knockdown of p27(kip1) with short hairpin RNA-expressing vectors results in the cell-cycle reentry of post-mitotic supporting cells in the post-natal mouse cochleae ex vivo. The p27(kip1)-knockdown cells incorporated BrdU, and then divided into two daughter cells. However, there was also activation of the apoptotic pathway in some supporting cells. These results indicate that the use of RNA interference to target p27(kip1) is an effective strategy for inducing cell-cycle reentry in post-mitotic supporting cells in the post-natal mammalian cochleae, although additional manipulations of the supporting cells are required to achieve hair cell regeneration.

Hepatocyte growth factor protects auditory hair cells from aminoglycosides.

OBJECTIVES/HYPOTHESIS: To examine the effect of hepatocyte growth factor (HGF) for protection of auditory hair cells against aminoglycosides and its molecular mechanisms. STUDY DESIGN: Experimental study. METHODS: We quantitatively assessed protective effects of HGF on mouse cochlear hair cells against neomycin toxicity using explant culture systems. To understand mechanisms of hair cell protection by HGF, we examined the expression of c-Met, HGF receptor, and 4-hydroxynonenal (a lipid peroxidation marker) in the cochlea by means of immunohistochemistry and Western blotting. RESULTS: The application of HGF to cochlear explant cultures significantly reduced the hair cell loss induced by neomycin. Immunohistochemistry showed c-Met expression in normal auditory hair cells, and its increase in response to neomycin-induced damage. Immunostaining for 4-hydroxynonenal suggested that HGF acted by attenuating the lipid peroxidation of auditory epithelia induced by neomycin. CONCLUSIONS: These findings demonstrate that a functional HGF/c-Met coupling is present in the cochlea, and HGF application exerts protective effects on hair cells, indicating the potential of HGF as a therapeutic agent for sensorineural hearing loss.

Transplantation of mouse induced pluripotent stem cells into the cochlea.

This study examined the potential of induced pluripotent stem (iPS) cells for use as a source of transplants for the restoration of auditory spiral ganglion neurons. We monitored neurite outgrowth from iPS cell-derived neural progenitors toward cochlear hair cells ex vivo, and followed their survival and fates after transplantation into mouse cochleae in vivo. Neurons derived from iPS cells projected neurites toward cochlear hair cells. The settlement of iPS cell-derived neurons was observed 1 week after transplantation into the cochlea. Some transplants expressed vesicular glutamate transporter 1, which is a marker for glutamatergic neurons. These findings indicate that iPS cells can be used as a source of transplants for the regeneration of spiral ganglion neurons.