Clarin-2 is essential for hearing by maintaining stereocilia integrity and function.

Hearing relies on mechanically gated ion channels present in the actin-rich stereocilia bundles at the apical surface of cochlear hair cells. Our knowledge of the mechanisms underlying the formation and maintenance of the sound-receptive structure is limited. Utilizing a large-scale forward genetic screen in mice, genome mapping and gene complementation tests, we identified Clrn2 as a new deafness gene. The Clrn2clarinet/clarinet mice (p.Trp4* mutation) exhibit a progressive, early-onset hearing loss, with no overt retinal deficits. Utilizing data from the UK Biobank study, we could show that CLRN2 is involved in human non-syndromic progressive hearing loss. Our in-depth morphological, molecular and functional investigations establish that while it is not required for initial formation of cochlear sensory hair cell stereocilia bundles, clarin-2 is critical for maintaining normal bundle integrity and functioning. In the differentiating hair bundles, lack of clarin-2 leads to loss of mechano-electrical transduction, followed by selective progressive loss of the transducing stereocilia. Together, our findings demonstrate a key role for clarin-2 in mammalian hearing, providing insights into the interplay between mechano-electrical transduction and stereocilia maintenance.

Key Characteristics for Successful Adoption and Implementation of Home Telehealth Technology in Veterans Affairs Home-Based Primary Care: An Exploratory Study.

BACKGROUND/OBJECTIVES: The Department of Veteran Affairs (VA) Home-Based Primary Care (HBPC) program provides care to over 37,000 high-risk, high-need, medically complex, and costly patients in their home. The VA's Home Telehealth (HT) program can potentially amplify HBPC's efficiency and reach, yet scarce data on use and experience with HT in HBPC exist. This exploratory study sought to provide a glimpse of HT use in HBPC and identify drivers and barriers for HT implementation. DESIGN: National VA data were used to evaluate HBPC patients concurrently using HT. We conducted a cross-sectional survey of HBPC program directors to explore HT use, understand communication processes, and elicit open comments. Semistructured interviews were conducted of 18 HBPC program directors with varying HT use to clarify themes and understand HBPC experience with HT. RESULTS: Fifteen percent of the overall HBPC patients used HT in 2011, with a wide variation in HT use by HBPC site. The national survey and semistructured interviews revealed that most HBPC staff recognized advantages of using HT, including increased patient engagement and staff efficiency. Crucial practices among sites with successful telehealth adoption included HT staff attending HBPC meetings and evaluating all HBPC patients for HT. CONCLUSION: Much remains to be done for effective HT integration in HBPC. Improving communication between HT and HBPC programs and establishing a system for identifying suitable patients for HT are vital. Future studies need to delineate operational processes and gather data on the added value of HT in HBPC to guide evidence-based integration of HT in VA and Medicare HBPC programs.

Novel gene function revealed by mouse mutagenesis screens for models of age-related disease.

Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss.

Absence of Neuroplastin-65 Affects Synaptogenesis in Mouse Inner Hair Cells and Causes Profound Hearing Loss.

The Neuroplastin gene encodes two synapse-enriched protein isoforms, Np55 and Np65, which are transmembrane glycoproteins that regulate several cellular processes, including the genesis, maintenance, and plasticity of synapses. We found that an absence of Np65 causes early-onset sensorineural hearing loss and prevented the normal synaptogenesis in inner hair cells (IHCs) in the newly identified mouse mutant pitch. In wild-type mice, Np65 is strongly upregulated in the cochlea from around postnatal day 12 (P12), which corresponds to the onset of hearing. Np65 was specifically localized at the presynaptic region of IHCs. We found that the colocalization of presynaptic IHC ribbons and postsynaptic afferent terminals is greatly reduced in pitch mutants. Moreover, IHC exocytosis is also reduced with mutant mice showing lower rates of vesicle release. Np65 appears to have a nonessential role in vision. We propose that Np65, by regulating IHC synaptogenesis, is critical for auditory function in mammals. SIGNIFICANCE STATEMENT: In the mammalian cochlea, the sensory inner hair cells (IHCs) encode auditory information. They do this by converting sound wave-induced mechanical motion of their hair bundles into an electrical current. This current generates a receptor potential that controls release of glutamate neurotransmitter from their ribbon synapses onto the auditory afferent fiber. We show that the synapse-enriched protein Np65, encoded by the Neuroplastin gene, is localized at the IHC presynaptic region. In mutant mice, absence of Np65 causes early-onset sensorineural hearing loss and prevents normal neurotransmitter release in IHCs and colocalization of presynaptic ribbons with postsynaptic afferents. We identified Neuroplastin as a novel deafness gene required for ribbon synapse formation and function, which is critical for sound perception in mammals.

The goya mouse mutant reveals distinct newly identified roles for MAP3K1 in the development and survival of cochlear sensory hair cells.

Mitogen-activated protein kinase, MAP3K1, plays an important role in a number of cellular processes, including epithelial migration during eye organogenesis. In addition, studies in keratinocytes indicate that MAP3K1 signalling through JNK is important for actin stress fibre formation and cell migration. However, MAP3K1 can also act independently of JNK in the regulation of cell proliferation and apoptosis. We have identified a mouse mutant, goya, which exhibits the eyes-open-at-birth and microphthalmia phenotypes. In addition, these mice also have hearing loss. The goya mice carry a splice site mutation in the Map3k1 gene. We show that goya and kinase-deficient Map3k1 homozygotes initially develop supernumerary cochlear outer hair cells (OHCs) that subsequently degenerate, and a progressive profound hearing loss is observed by 9 weeks of age. Heterozygote mice also develop supernumerary OHCs, but no cellular degeneration or hearing loss is observed. MAP3K1 is expressed in a number of inner-ear cell types, including outer and inner hair cells, stria vascularis and spiral ganglion. Investigation of targets downstream of MAP3K1 identified an increase in p38 phosphorylation (Thr180/Tyr182) in multiple cochlear tissues. We also show that the extra OHCs do not arise from aberrant control of proliferation via p27KIP1. The identification of the goya mutant reveals a signalling molecule involved with hair-cell development and survival. Mammalian hair cells do not have the ability to regenerate after damage, which can lead to irreversible sensorineural hearing loss. Given the observed goya phenotype, and the many diverse cellular processes that MAP3K1 is known to act upon, further investigation of this model might help to elaborate upon the mechanisms underlying sensory hair cell specification, and pathways important for their survival. In addition, MAP3K1 is revealed as a new candidate gene for human sensorineural hearing loss.

Interactions between the otitis media gene, Fbxo11, and p53 in the mouse embryonic lung.

Otitis media with effusion (OME) is the most common cause of hearing loss in children, and tympanostomy (ear tube insertion) to alleviate the condition remains the commonest surgical intervention in children in the developed world. Chronic and recurrent forms of otitis media (OM) are known to have a very substantial genetic component; however, until recently, little was known of the underlying genes involved. The Jeff mouse mutant carries a mutation in the Fbxo11 gene, a member of the F-box family, and develops deafness due to a chronic proliferative OM. We previously reported that Fbxo11 is involved in the regulation of transforming growth factor beta (TGF-ß) signalling by regulating the levels of phospho-Smad2 in the epithelial cells of palatal shelves, eyelids and airways of the lungs. It has been proposed that FBXO11 regulates the cell's response to TGF-ß through the ubiquitination of CDT2. Additional substrates for FBXO11 have been identified, including p53. Here, we have studied both the genetic and biochemical interactions between FBXO11 and p53 in order to better understand the function of FBXO11 in epithelial development and its potential role in OM. In mice, we show that p53 (also known as Tp53) homozygous mutants and double heterozygous mutants (Jf/+ p53/+) exhibit similar epithelial developmental defects to Fbxo11 homozygotes. FBXO11 and p53 interact in the embryonic lung, and mutation in Fbxo11 prevents the interaction with p53. Both p53 and double mutants show raised levels of pSMAD2, recapitulating that seen in Fbxo11 homozygotes. Overall, our results support the conclusion that FBXO11 regulates the TGF-ß pathway in the embryonic lung via cross-talk with p53.

Otitis media in the Tgif knockout mouse implicates TGFß signalling in chronic middle ear inflammatory disease.

Otitis media with effusion (OME) is the most common cause of hearing loss in children and tympanostomy to alleviate the condition remains the commonest surgical intervention in children in the developed world. Chronic and recurrent forms of OM are known to have a very significant genetic component, however, until recently little was known of the underlying genes involved. The identification of mouse models of chronic OM has indicated a role of transforming growth factor beta (TGFß) signalling and its impact on responses to hypoxia in the inflamed middle ear. We have, therefore, investigated the role of TGFß signalling and identified and characterized a new model of chronic OM carrying a mutation in the gene for transforming growth interacting factor 1 (Tgif1). Tgif1 homozygous mutant mice have significantly raised auditory thresholds due to a conductive deafness arising from a chronic effusion starting at around 3 weeks of age. The OM is accompanied by a significant thickening of the middle ear mucosa lining, expansion of mucin-secreting goblet cell populations and raised levels of vascular endothelial growth factor, TNF-α and IL-1ß in ear fluids. We also identified downstream effects on TGFß signalling in middle ear epithelia at the time of development of chronic OM. Both phosphorylated SMAD2 and p21 levels were lowered in the homozygous mutant, demonstrating a suppression of the TGFß pathway. The identification and characterization of the Tgif mutant supports the role of TGFß signalling in the development of chronic OM and provides an important candidate gene for genetic studies in the human population.

Regulation of TGF-beta signalling by Fbxo11, the gene mutated in the Jeff otitis media mouse mutant.

BACKGROUND: Jeff is a dominant mouse mutant displaying chronic otitis media. The gene underlying Jeff is Fbxo11, a member of the large F-box family, which are specificity factors for the SCF E3 ubiquitin ligase complex. Jeff homozygotes die shortly after birth displaying a number of developmental abnormalities including cleft palate and eyes open at birth. TGF-beta signalling is involved in a number of epithelial developmental processes and we have investigated the impact of the Jeff mutation on the expression of this pathway. RESULTS: Phospho-Smad2 (pSmad2) is significantly upregulated in epithelia of Jeff homozygotes. Moreover, there was a significant increase in nuclear localization of pSmad2 in contrast to wild type. Mice heterozygous for both Jeff and Smad2 mutations recapitulate many of the features of the Jeff homozygous phenotype. However, tissue immunoprecipitations failed to detect any interaction between Fbxo11 and Smad2. Fbxo11 is known to neddylate p53, a co-factor of pSmad2, but we did not find any evidence of genetic interactions between Jeff and p53 mutants. Nevertheless, p53 levels are substantially reduced in Jeff mice suggesting that Fbxo11 plays a role in stabilizing p53. CONCLUSION: Overall, our findings support a model whereby Fbxo11, possibly via stabilization of p53, is required to limit the accumulation of pSmad2 in the nucleus of epithelial cells of palatal shelves, eyelids and airways of the lungs. The finding that Fbxo11 impacts upon TGF-beta signalling has important implications for our understanding of the underlying disease mechanisms of middle ear inflammatory disease.

A mutation in the F-box gene, Fbxo11, causes otitis media in the Jeff mouse.

Otitis media (OM), inflammation of the middle ear, is the most common cause of hearing impairment and surgery in children. Recurrent and chronic forms of OM are known to have a strong genetic component, but nothing is known of the underlying genes involved in the human population. We have previously identified a novel semi-dominant mouse mutant, Jeff, in which the heterozygotes develop chronic suppurative OM (Hardisty, R.E., Erven, A., Logan, K., Morse, S., Guionaud, S., Sancho-Oliver, S., Hunter, A.J., Brown, S.D. and Steel, K.P. (2003) The deaf mouse mutant Jeff (Jf) is a single gene model of otitis media. J. Assoc. Res. Otolaryngol., 4, 130-138.) and represent a model for chronic forms of OM in humans. We demonstrate here that Jeff carries a mutation in an F-box gene, Fbxo11. Fbxo11 is expressed in epithelial cells of the middle ears from late embryonic stages through to day 13 of postnatal life. In contrast to Jeff heterozygotes, Jeff homozygotes show cleft palate, facial clefting and perinatal lethality. We have also isolated and characterized an additional hypomorphic mutant allele, Mutt. Mutt heterozygotes do not develop OM but Mutt homozygotes also show facial clefting and cleft palate abnormalities. FBXO11 is one of the first molecules to be identified, contributing to the genetic aetiology of OM. In addition, the recessive effects of mutant alleles of Fbxo11 identify the gene as an important candidate for cleft palate studies in the human population.

The deaf mouse mutant Jeff (Jf) is a single gene model of otitis media.

Otitis media is the most common cause of hearing impairment in children and is primarily characterized by inflammation of the middle ear mucosa. Yet nothing is known of the underlying genetic pathways predisposing to otitis media in the human population. Increasingly, large-scale mouse mutagenesis programs have undertaken systematic and genome-wide efforts to recover large numbers of novel mutations affecting a diverse array of phenotypic areas involved with genetic disease including deafness. As part of the UK mutagenesis program, we have identified a novel deaf mouse mutant, Jeff (Jf). Jeff maps to the distal region of mouse chromosome 17 and presents with fluid and pus in the middle ear cavity. Jeff mutants are 21% smaller than wild-type littermates, have a mild craniofacial abnormality, and have elevated hearing thresholds. Middle ear epithelia of Jeff mice show evidence of a chronic proliferative otitis media. The Jeff mutant should prove valuable in elucidating the underlying genetic pathways predisposing to otitis media.