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Multiple mouse embryonic stem (ES) cell banks expand the capability to characterize functions of genes implicated in human disease and to develop mouse models for the further understanding of disease pathology. Genetic diseases that result in hearing loss can provide insight into causative molecular mechanisms for deafness. We utilized BayGenomics, the public mouse ES cell bank, to identify gene-trapped ES cell lines associated with hearing loss. We identified two gene-trapped ES cell lines specific for the non-muscle myosin heavy chain class IIA or myosin heavy chain IX (Myh9). Inherited mutations in the Myh9 gene have been linked to non-syndromic hereditary hearing impairment DFNA17 as well as 'MYH9-related disease' characterized by macrothrombocytopenia, leukocyte inclusions, and in some patients deafness. Mutant Myh9 mice were derived from one of these ES cell lines that underwent germline transmission for in-depth otological examination. No homozygous mice however were identified at birth, consistent with recently published data describing the embryonic lethality of homozygous mutations in Myh9. We provide evidence that adult heterozygous Myh9 mouse inner ears contain half wild-type levels of Myh9 mRNA. Hearing loss however was not observed in heterozygous Myh9 mice in contrast to human Myh9-related diseases. Aged heterozygous Myh9 mice also did not show signs of cochleosaccular degeneration common in DFNA17. Although inheritance of Myh9 mutations in humans is dominant, we conclude that heterozygous loss of Myh9 is not critical to hearing function in mice by itself.

Multiple PDZ domain protein 1 (MUPP1), a putative scaffolding protein containing 13 PSD-95, Dlg, ZO-1 (PDZ) domains, was identified by a yeast two-hybrid screen as a serotonin2C receptor (5-HT2C R)-interacting protein (Ullmer, C., Schmuck, K., Figge, A., and Lubbert, H. (1998) FEBS Lett. 424, 63-68). MUPP1 PDZ domain 10 (PDZ 10) associates with Ser458-Ser-Val at the carboxyl-terminal tail of the 5-HT2C R. Both Ser458 and Ser459 are phosphorylated upon serotonin stimulation of the receptor (Backstrom, J. R., Price, R. D., Reasoner, D. T., and Sanders-Bush, E. (2000) J. Biol. Chem. 275, 23620-23626). To investigate whether phosphorylation of these serines in the receptor regulates MUPP1 interaction, we used several approaches. First, we substituted the serines in the receptor carboxyl tail with aspartates to mimic phosphorylation (S458D, S459D, or S458D/S459D). Pull-down assays demonstrated that Asp mutations at Ser458 significantly decreased receptor tail interaction with PDZ 10. Next, serotonin treatment of 5-HT2C R/3T3 cells resulted in a dose-dependent reduction of receptor interaction with PDZ 10. Effects of serotonin on receptor-PDZ 10 binding could be blocked by pretreatment with a receptor antagonist. Alkaline phosphatase treatment reverses the effect of serotonin, indicating that agonist-induced phosphorylation at Ser458 resulted in a loss of MUPP1 association and also revealed a significant amount of basal phosphorylation of the receptor. We conclude that 5-HT2C R interaction with MUPP1 is dynamically regulated by phosphorylation at Ser458.

Cochlear hair cells express SK2, a small-conductance Ca2+-activated K+ channel thought to act in concert with Ca2+-permeable nicotinic acetylcholine receptors (nAChRs) α9 and α10 in mediating suppressive effects of the olivocochlear efferent innervation. To probe the in vivo role of SK2 channels in hearing, we examined gene expression, cochlear function, efferent suppression, and noise vulnerability in mice overexpressing SK2 channels. Cochlear thresholds, as measured by auditory brain stem responses and otoacoustic emissions, were normal in overexpressers as was overall cochlear morphology and the size, number, and distribution of efferent terminals on outer hair cells. Cochlear expression levels of SK2 channels were elevated eightfold without striking changes in other SK channels or in the α9/α10 nAChRs. Shock-evoked efferent suppression of cochlear responses was significantly enhanced in overexpresser mice as seen previously in α9 overexpresser mice; however, in contrast to α9 overexpressers, SK2 overexpressers were not protected from acoustic injury. Results suggest that efferent-mediated cochlear protection is mediated by other downstream effects of ACh-mediated Ca2+ entry different from those involving SK2-mediated hyperpolarization and the associated reduction in outer hair cell electromotility.

The Tennessee Mouse Genome Consortium (TMGC) employed an N-ethyl-N-nitrosourea (ENU)-mutagenesis scheme to identify mouse recessive mutants with hearing phenotypes. We employed auditory brainstem responses (ABR) to click and 8, 16, and 32 kHz stimuli and screened 285 pedigrees (1819 mice of 8-11 weeks old in various mixed genetic backgrounds) each bred to carry a homozygous ENU-induced mutation. To define mutant pedigrees, we measured P12 mice per pedigree in P2 generations and used a criterion where the mean ABR threshold per pedigree was two standard deviations above the mean of all offspring from the same parental strain. We thus identified 17 mutant pedigrees (6%), all exhibiting hearing loss at high frequencies (P16 kHz) with an average threshold elevation of 30-35 dB SPL. Interestingly, four mutants showed sex-biased hearing loss and six mutants displayed wide range frequency hearing loss. Temporal bone histology revealed that six of the first nine mutants displayed cochlear morphological defects: degeneration of spiral ganglia, spiral ligament fibrocytes or inner hair cells (but not outer hair cells) mostly in basal turns. In contrast to other ENU-mutagenesis auditory screens, our screen identified high-frequency, mild and sex-biased hearing defects. Further characterization of these 17 mouse models will advance our understanding ofmore » presbycusis and noise-induced hearing loss in humans.« less