Adult-onset hearing loss is very common, but we know little about the underlying molecular pathogenesis impeding the development of therapies. We took a genetic approach to identify new molecules involved in hearing loss by screening a large cohort of newly generated mouse mutants using a sensitive electrophysiological test, the auditory brainstem response (ABR). We review here the findings from this screen. Thirty-eight unexpected genes associated with raised thresholds were detected from our unbiased sample of 1,211 genes tested, suggesting extreme genetic heterogeneity. A wide range of auditory pathophysiologies was found, and some mutant lines showed normal development followed by deterioration of responses, revealing new molecular pathways involved in progressive hearing loss. Several of the genes were associated with the range of hearing thresholds in the human population and one, SPNS2 , was involved in childhood deafness. The new pathways required for maintenance of hearing discovered by this screen present new therapeutic opportunities.

In the mammalian inner ear, bicellular and tricellular tight junctions (tTJs) seal the paracellular space between epithelial cells. Tricellulin and immunoglobulin-like (Ig-like) domain containing receptor 1 (ILDR1, also referred to as angulin-2) localize to tTJs of the sensory and non-sensory epithelia in the organ of Corti and vestibular end organs. Recessive mutations of TRIC (DFNB49) encoding tricellulin and ILDR1 (DFNB42) cause human nonsyndromic deafness. However, the pathophysiology of DFNB42 deafness remains unknown. ILDR1 was recently reported to be a lipoprotein receptor mediating the secretion of the fat-stimulated cholecystokinin (CCK) hormone in the small intestine, while ILDR1 in EpH4 mouse mammary epithelial cells in vitro was shown to recruit tricellulin to tTJs. Here we show that two different mouse Ildr1 mutant alleles have early-onset severe deafness associated with a rapid degeneration of cochlear hair cells (HCs) but have a normal endocochlear potential. ILDR1 is not required for recruitment of tricellulin to tTJs in the cochlea in vivo; however, tricellulin becomes mislocalized in the inner ear sensory epithelia of ILDR1 null mice after the first postnatal week. As revealed by freeze-fracture electron microscopy, ILDR1 contributes to the ultrastructure of inner ear tTJs. Taken together, our data provide insight into the pathophysiology of human DFNB42 deafness and demonstrate that ILDR1 is crucial for normal hearing by maintaining the structural and functional integrity of tTJs, which are critical for the survival of auditory neurosensory HCs.

These results identify the reversible acetylation of Notch1 as a novel mechanism to modulate its signalling in the heart and tune the proliferative potential of cardiomyocytes.

Progressive hearing loss is very common in the human population but we know little about the underlying molecular mechanisms. Synaptojanin2 (Synj2) has been reported to be involved, as a mouse mutation led to a progressive increase in auditory thresholds with age. Synaptojanin2 is a phosphatidylinositol (PI) phosphatase that removes the five-position phosphates from phosphoinositides, such as PIP 2 and PIP 3 , and is a key enzyme in clathrin-mediated endocytosis. To investigate the mechanisms underlying progressive hearing loss, we have studied a different mutation of mouse Synj2 to look for any evidence of involvement of vesicle trafficking particularly affecting the synapses of sensory hair cells. Auditory brainstem responses (ABR) developed normally at first but started to decline between 3 and 4 weeks of age in Synj2 tm1b mutants. At 6 weeks old, some evidence of outer hair cell (OHC) stereocilia fusion and degeneration was observed, but this was only seen in the extreme basal turn so cannot explain the raised ABR thresholds that correspond to more apical regions of the cochlear duct. We found no evidence of any defect in inner hair cell (IHC) exocytosis or endocytosis using single hair cell recordings, nor any sign of hair cell synaptic abnormalities. Endocochlear potentials (EP) were normal. The mechanism underlying progressive hearing loss in these mutants remains elusive, but our findings of raised distortion product otoacoustic emission (DPOAE) thresholds and signs of OHC degeneration both suggest an OHC origin for the hearing loss. Synaptojanin2 is not required for normal development of hearing but it is important for its maintenance.

Peroxisome biogenesis disorders (due to PEX gene mutations) are associated with symptoms that range in severity and can lead to early childhood death, but a common feature is hearing impairment. In this study, mice carrying Pex3 mutations were found to show normal auditory development followed by an early-onset progressive increase in auditory response thresholds. The only structural defect detected in the cochlea at four weeks old was the disruption of synapses below inner hair cells. A conditional approach was used to establish that Pex3 expression is required locally within the cochlea for normal hearing, rather than hearing loss being due to systemic effects. A lipidomics analysis of the inner ear revealed a local reduction in plasmalogens in the Pex3 mouse mutants, comparable to the systemic plasmalogen reduction reported in human peroxisome biogenesis disorders. Thus, mice with Pex3 mutations may be a useful tool to understand the physiological basis of peroxisome biogenesis disorders.

Hearing loss is highly heterogeneous, but one common form involves a failure to maintain the local ionic environment of the sensory hair cells reflected in a reduced endocochlear potential. We used a genetic approach to ask whether this type of pathology can be reversed, using the Spns2 tm1a mouse mutant known to show this defect. By activating Spns2 gene transcription at different ages after the onset of hearing loss, we found that an existing auditory impairment can be reversed to give close to normal thresholds for an auditory brainstem response (ABR), at least at low to mid stimulus frequencies. Delaying the activation of Spns2 led to less effective recovery of ABR thresholds, suggesting that there is a critical period for intervention. Early activation of Spns2 not only led to improvement in auditory function but also to protection of sensory hair cells from secondary degeneration. The genetic approach we have used to establish that this type of hearing loss is in principle reversible could be extended to many other diseases using available mouse resources.

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