My oh my(osin): Insights into how auditory hair cells count,measure, and shape

The mechanisms underlying mechanosensory hair bundle formation in auditory sensory cells are largely mysterious. In this issue, Lelli et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201509017) reveal that a pair of molecular motors, myosin IIIa and myosin IIIb, is involved in the hair bundle’s morphology and hearing.The mammalian cochleae are lined with hair cells, each with a precisely arranged, morphologically polar hair bundle that captures energy produced by sound stimuli. A fascinating problem in cell biology is how this elaborately structured mechanosensory hair bundle forms with precision to enable hearing. Each hair bundle is comprised of up to a few hundred actin-filled stereocilia, arrayed in a staircase-like pattern of increasing heights. Deflection of a hair bundle in the direction of the tallest stereocilium opens mechanically gated ion channels at the stereociliary tips, allowing an influx of cations from the endolymph bathing the stereocilia and thus depolarizing the cell. The staircase array of stereocilia forms by elongation of microvilli at the apical surface of the developing hair cell (Tilney et al., 1992). Over 20 years ago, Tilney et al. (1992) detailed the steps of stereociliogenesis as observed by electron microscopy. In this issue, Lelli et al. provide molecular insight into how hair cells count, measure, and shape stereocilia.Loss of hair cells is a major cause of human hearing loss, which is often the result of genetic mutations affecting the development of stereocilia (Raphael, 2002). Mutations in genes encoding several different myosin motor proteins in stereocilia have been associated with human hearing loss, including myosin VIIA (Weil et al., 1995), myosin VI (Avraham et al., 1995; Melchionda et al., 2001), and myosin XVA (Wang et al., 1998). Additionally, loss-of-function mutations in MYO3A, encoding myosin IIIA, are responsible for hereditary progressive hearing loss DFNB30 (Walsh et al., 2002).Myosins, the ATP-dependent motors that move along actin-based filaments, are typically composed of three functional domains: the head, the neck, and the tail domains (Krendel and Mooseker, 2005). Class III myosins are unconventional myosins that each have a kinase domain at their N terminus regulated by PKA phosphorylation and autophosphorylation (Kempler et al., 2007). The kinase activity of myosin III was shown to act on its own motor domain to reduce the motor activity. Myosin III proteins concentrate at actin-based cellular protrusions, such as stereocilia of inner ear hair cells (Dosé et al., 2003; Schneider et al., 2006). The two class III isoforms found in vertebrates, myosin IIIa and IIIb, differ toward their C termini: myosin IIIa is longer than myosin IIIb and has one additional actin-binding domain, myosin III tail homology domain II (3THDII). In cultured cells, whereas myosin IIIa localizes to the tips of filopodia on its own, previous work showed that myosin IIIb requires its interaction partner espin-1, an actin-binding protein, for localization to filopodia tips (Merritt et al., 2012). Mutagenesis studies performed on myosin IIIa revealed a relationship between the phosphorylation state of myosin IIIa and the length and density of filopodia (Quintero et al., 2013). How myosin IIIa and IIIb work individually or together to regulate stereociliogenesis remains to be investigated.In this issue, Lelli et al. (2016) use Myo3a (Myo3a^−/−), Myo3b (Myo3b^−/−), and double (Myo3a^−/−Myo3b^−/−) knockout mice to dissect the complex roles of myosin IIIa and IIIb in hearing. Mice null for myosin IIIa, Myo3a^−/−, were initially normal but showed defects in hearing quality from 2 to 4 mo of age, as detected by auditory brain stem response measurements, which monitor the electrical response of the auditory pathway to short sound stimuli, but not by distortion product otoacoustic emissions, which test outer hair cell (OHC) function. These results indicated that inner hair cells (IHCs) are deleteriously impacted but that OHCs are not, and that these mice had progressive hearing loss. Myo3b^−/− mutants had normal hearing, indicating that redundancy with myosin IIIa may obscure the role of this protein in single knockouts. Investigation of double knockouts, however, clarified the redundant functions of the two isoforms, as the mice were profoundly deaf at 1 mo of age.Interestingly, Lelli et al. (2016) investigated the potential requirement for myosin IIIa in the maintenance of hearing. They generated conditional knockout (cKO) mice, Myo3a-cKO, in which myosin IIIa is inactivated postnatally in a Myo3b^−/− background. These animals hear normally up to at least 6 mo of age. Therefore, myosin IIIa is required during a critical period in auditory system development but is not required to maintain hearing. The researchers further studied the relationship between myosin IIIa and myosin IIIb in the Myo3a-cKO mice and noted an increase in auditory brain stem response thresholds. This fascinating result likely indicates that inactivation of myosin IIIa elicits a pernicious effect of myosin IIIb at mature stages. Determining the mechanism by which myosin IIIb damages hearing in the absence of myosin IIIa and whether it is due to its lack of a single 3THDII domain may yield insight into the roles of class III myosins in hearing.Given that myosin III proteins localize to the tips of stereocilia and are required for hearing, Lelli et al. (2016) explored the morphology of hair bundles lacking these proteins. Although double knockout Myo3a^−/−Myo3b^−/− mice displayed normal positioning of the kinocilium, the cilium reflecting the initial position and polarity of the developing hair bundle, and of asymmetric cell division proteins Par-6 and Gαi₃, which constrain the shape and positioning of the hair bundle, many hair cells from these mice exhibited hair bundle abnormalities. These abnormalities were first described during embryonic auditory hair bundle morphogenesis, at which time 19% of IHCs and 81% of OHCs displayed misshapen bundles. Several of these misshapen IHC and OHC bundles also contained abnormally long, ungraded protrusions, which Lelli et al. (2016) referred to as “long amorphous” bundles. By birth, most IHCs displayed long amorphous bundles. Although most OHC hair bundles were abnormally shaped, they had developed normal staircase organization of their stereocilia heights, and the long amorphous protrusions were no longer present. Presumably, another molecular mechanism comes into play and “corrects” the protrusions to establish the staircase organization. Knockout of candidate proteins could be an interesting strategy to uncover this mechanism in the future. Furthermore, other stereociliary phenotypes of Myo3a^−/−Myo3b^−/− mutant mice are also intriguing. Do myosin III proteins serve different mechanistic roles in IHCs and OHCs to give rise to different phenotypes? Or, are there other key factors that drive stereocilia into various degrees of hair bundle defects in the absence of myosin III proteins?In Myo3a^−/−Myo3b^−/− mice, many of the OHC bundles had abnormal side rows of extra stereocilia, which sometimes closed the bundle off into a circular shape. Interestingly, the stereocilia in these abnormal bundles were also significantly taller than in controls. Although the height of the tallest row of stereocilia in OHCs normally decreases after birth (Sekerková et al., 2011), the height of these stereocilia did not change in the Myo3a^−/−Myo3b^−/− mice, leading to an increased height difference 9 d after birth. The increased height and number of stereocilia in Myo3a^−/−Myo3b^−/− hair bundles is suggestive of unstable actin dynamics. The authors suggest that class III myosins stabilize the F-actin cores of the stereocilia, thus controlling their selective elongation by limiting their growth (Fig. 1). Surprisingly, class III myosins, which presumably climb the full lengths of the stereocilia to perch themselves near the tips, act to restrict their growth. This result seems to contrast with previous work in which myosin IIIa had been proposed to promote elongation of the stereocilia by transporting espin-1 to the stereocilia tips (Salles et al., 2009). Interestingly, Lelli et al. (2016) found that espin-1 was still properly targeted to the tips of stereocilia in Myo3a^−/−Myo3b^−/− mutant mice. The researchers extended the study to another binding partner of myosin IIIa, retinophilin/MORN4. Surprisingly, MORN4 was normally targeted to stereocilia tips in Myo3a^−/−Myo3b^−/− mutant mice. This suggests that there may be redundancy in the mechanisms of transport of stereocilia tip proteins. Whether or not distinct myosin isoforms interchange cargoes so that these cargoes are properly localized is a relevant avenue of future investigation. Furthermore, identification and knockout of class III myosin binding partners could help define the mechanism by which these proteins control elongation of stereocilia.Open in a separate windowFigure 1.Myosin IIIa and myosin IIIb regulate stereociliary length. (top) Schematics of myosin IIIa and myosin IIIb. The stop signs indicate the role these proteins have in limiting the heights of stereocilia. (bottom) A single stereocilium with wild-type copies of myosin IIIa and myosin IIIb is shorter than a stereocilium from a Myo3a^−/−Myo3b^−/− mutant when stereocilia from equivalent positions between hair bundles are compared. In addition to characterizing the lengths of stereocilia, Lelli et al. (2016) noted transient defects in the numbers of stereocilia during development of hair cells within genetically altered mice. At birth, Myo3a^−/−Myo3b^−/− mice had an increased number and density of stereocilia in both IHCs and OHCs as compared with controls. However, 9 d after birth, the number and density of the stereocilia had decreased to levels similar to those seen in control animals. Their results indicate that class III myosins help to control the initial selection of microvilli to become stereocilia, but a different, dominant molecular mechanism overrides this step and is likely involved in refining the number of stereocilia at later, postnatal stages. Identification of this mechanism by knockout of candidate molecules in the Myo3a^−/−Myo3b^−/− mice would be of interest for future work. The defects, though transient, indicate that myosins are key molecular components that establish how cells count the number of protrusions to be produced.In this work, Lelli et al. (2016) determined that myosins IIIa and IIIb are required for normal hair bundle development and hearing. These motor proteins influence the number and lengths of stereocilia to be produced and the overarching shape of the hair bundle. Evaluating the importance of the kinase domains of myosin IIIa and IIIb in hair bundle development is an important next step. The phosphorylation states of proteins in cellular protrusions, such as filopodia and stereocilia, can be regulatory. For example, phosphorylation of the actin cross-linking protein fascin 2b reduces this protein’s capacity to lengthen filopodia (Chou et al., 2011). Moreover, phosphorylation of fascin 2b increases the exchange rate of this protein within zebrafish stereocilia (Hwang et al., 2015). Additionally, in other systems, PKA modulates a signaling cascade that activates the actin-severing protein cofilin to control actin-filament dynamics (Nadella et al., 2009). Future work will be needed to test whether signal transduction stemming from the kinase domains of class III myosins are consequential for hair bundle development.Lelli et al. (2016) observed that the constitutive absence of both class III myosins leads to deafness as a result of hair bundle developmental defects. However, mice deficient for only one of the isoforms did not display such a striking hearing phenotype, indicating that myosins IIIa and IIIb compensate for the loss of one another in the developing cochlea. A role for myosin IIIa in the maintenance of stereocilia had been suggested by the discovery of loss-of-function mutations in MYO3A in patients with hereditary progressive hearing loss DFNB30 (Walsh et al., 2002). Remarkably, the analyses of Myo3a^−/−Myo3b^−/− and Myo3a-cKO Myo3b^−/− mice showed that the redundancy between myosins IIIa and IIIb is critical during hair bundle formation, but not during later, mature stages. Furthermore, the hearing impairment seen in Myo3a-cKO mice suggests that myosin IIIb exerts deleterious effects on hearing past the developmental stages of hair bundle morphogenesis. Although the mechanisms at work are unclear, these findings suggest that the down-regulation of MYO3B expression might be an effective way of preventing late-onset hearing loss in patients with MYO3A mutations.     

Compound Heterozygosity of the Functionally Null Cdh23v-ngt and Hypomorphic Cdh23ahl Alleles Leads to Early-onset Progressive Hearing Loss in Mice

The waltzer (v) mouse mutant harbors a mutation in Cadherin 23 (Cdh23) and is a model for Usher syndrome type 1D, which is characterized by congenital deafness, vestibular dysfunction, and prepubertal onset of progressive retinitis pigmentosa. In mice, functionally null Cdh23 mutations affect stereociliary morphogenesis and the polarity of both cochlear and vestibular hair cells. In contrast, the murine Cdh23^ahl allele, which harbors a hypomorphic mutation, causes an increase in susceptibility to age-related hearing loss in many inbred strains. We produced congenic mice by crossing mice carrying the v niigata (Cdh23^v-ngt) null allele with mice carrying the hypomorphic Cdh23^ahl allele on the C57BL/6J background, and we then analyzed the animals’ balance and hearing phenotypes. Although the Cdh23^v-ngt/ahl compound heterozygous mice exhibited normal vestibular function, their hearing ability was abnormal: the mice exhibited higher thresholds of auditory brainstem response (ABR) and rapid age-dependent elevation of ABR thresholds compared with Cdh23^ahl/ahl homozygous mice. We found that the stereocilia developed normally but were progressively disrupted in Cdh23^v-ngt/ahl mice. In hair cells, CDH23 localizes to the tip links of stereocilia, which are thought to gate the mechanoelectrical transduction channels in hair cells. We hypothesize that the reduction of Cdh23 gene dosage in Cdh23^v-ngt/ahl mice leads to the degeneration of stereocilia, which consequently reduces tip link tension. These findings indicate that CDH23 plays an important role in the maintenance of tip links during the aging process.     

Correction: Talin tension sensor reveals novel features of focal adhesion force transmission and mechanosensitivity

The precise architecture of hair bundles, the arrays of mechanosensitive microvilli-like stereocilia crowning the auditory hair cells, is essential to hearing. Myosin IIIa, defective in the late-onset deafness form DFNB30, has been proposed to transport espin-1 to the tips of stereocilia, thereby promoting their elongation. We show that Myo3a^−/−Myo3b^−/− mice lacking myosin IIIa and myosin IIIb are profoundly deaf, whereas Myo3a-cKO Myo3b^−/− mice lacking myosin IIIb and losing myosin IIIa postnatally have normal hearing. Myo3a^−/−Myo3b^−/− cochlear hair bundles display robust mechanoelectrical transduction currents with normal kinetics but show severe embryonic abnormalities whose features rapidly change. These include abnormally tall and numerous microvilli or stereocilia, ungraded stereocilia bundles, and bundle rounding and closure. Surprisingly, espin-1 is properly targeted to Myo3a^−/−Myo3b^−/− stereocilia tips. Our results uncover the critical role that class III myosins play redundantly in hair-bundle morphogenesis; they unexpectedly limit the elongation of stereocilia and of subsequently regressing microvilli, thus contributing to the early hair bundle shaping.     

Ankyrin Repeat Domain Protein 2 and Inhibitor of DNA Binding 3 Cooperatively Inhibit Myoblast Differentiation by Physical Interaction

Ankyrin repeat domain protein 2 (ANKRD2) translocates from the nucleus to the cytoplasm upon myogenic induction. Overexpression of ANKRD2 inhibits C2C12 myoblast differentiation. However, the mechanism by which ANKRD2 inhibits myoblast differentiation is unknown. We demonstrate that the primary myoblasts of mdm (muscular dystrophy with myositis) mice (pMB^mdm) overexpress ANKRD2 and ID3 (inhibitor of DNA binding 3) proteins and are unable to differentiate into myotubes upon myogenic induction. Although suppression of either ANKRD2 or ID3 induces myoblast differentiation in mdm mice, overexpression of ANKRD2 and inhibition of ID3 or vice versa is insufficient to inhibit myoblast differentiation in WT mice. We identified that ANKRD2 and ID3 cooperatively inhibit myoblast differentiation by physical interaction. Interestingly, although MyoD activates the Ankrd2 promoter in the skeletal muscles of wild-type mice, SREBP-1 (sterol regulatory element binding protein-1) activates the same promoter in the skeletal muscles of mdm mice, suggesting the differential regulation of Ankrd2. Overall, we uncovered a novel pathway in which SREBP-1/ANKRD2/ID3 activation inhibits myoblast differentiation, and we propose that this pathway acts as a critical determinant of the skeletal muscle developmental program.     

Stereocilium injury mediates hair bundle stiffness loss and recovery following intense water-jet stimulation

Inner ear hair cells exhibit many pathologies following exposure to intense sound, and the hair bundle is a major site of damage. This paper measures in vitro hair bundle motion on chick cochlear hair cells after intense in vitro and in vivo stimulation to explore the nature of hair bundle injury. Hair bundle stiffness, as well as relative and asymmetric motion of individual stereocilia, is controlled largely by the extracellular tip links, and a change in hair bundle motion was used to assess tip-link destruction following overstimulation. Intense in vitro stimulation caused a loss in stiffness that fully recovered within 10 min post-exposure. Relative and asymmetric stereocilia motion, however, were unchanged following the exposure, implying that tip links remained intact while the core or rootlet of the stereocilia were damaged and subsequently repaired. Intense and prolonged in vivo sound exposures produced stereocilia movements, measured in vitro, that were indicative of damage to stereocilia and tip links. Finally, the relative susceptibility of hair bundles to overstimulation was addressed by comparing stiffness loss with morphological features in the hair bundles. The loss of stiffness significantly increased as the amount of curvature in the hair bundle contour increased.     

Transient Receptor Potential Melastatin 1: A Hair Cell Transduction Channel Candidate

Sound and head movements are perceived through sensory hair cells in the inner ear. Mounting evidence indicates that this process is initiated by the opening of mechanically sensitive calcium-permeable channels, also referred to as the mechanoelectrical transducer (MET) channels, reported to be around the tips of all but the tallest stereocilia. However, the identity of MET channel remains elusive. Literature suggests that the MET channel is a non-selective cation channel with a high Ca²⁺ permeability and ∼100 picosiemens conductance. These characteristics make members of the transient receptor potential (TRP) superfamily likely candidates for this role. One of these candidates is the transient receptor potential melastatin 1 protein (TRPM1), which is expressed in various cells types within the cochlea of the mouse including the hair cells. Recent studies demonstrate that mutations in the TRPM1 gene underlie the inherited retinal disease complete congenital stationary night blindness in humans and depolarizing bipolar cell dysfunction in the mouse retina, but auditory function was not assessed. Here we investigate the role of Trpm1 in hearing and as a possible hair cell MET channel using mice homozygous for the null allele of Trpm1 (Trpm1^−/−) or a missense mutation in the pore domain of TRPM1 (Trpm1^{tvrm27/tvrm27}). Hearing thresholds were evaluated in adult (4–5 months old) mice with auditory-evoked brain stem responses. Our data shows no statistically significant difference in hearing thresholds in Trpm1^−/− or Trpm1^{tvrm27/tvrm27} mutants compared to littermate controls. Further, none of the mutant mice showed any sign of balance disorder, such as head bobbing or circling. These data suggest that TRPM1 is not essential for development of hearing or balance and it is unlikely that TRPM1 is a component of the hair cell MET channel.     

Reduction in Noise-Induced Functional Loss of the Cochleae in Mice with Pre-Existing Cochlear Dysfunction Due to Genetic Interference of Prestin

Various cochlear pathologies, such as acoustic trauma, ototoxicity and age-related degeneration, cause hearing loss. These pre-existing hearing losses can alter cochlear responses to subsequent acoustic overstimulation. So far, the knowledge on the impacts of pre-existing hearing loss caused by genetic alteration of cochlear genes is limited. Prestin is the motor protein expressed exclusively in outer hair cells in the mammalian cochlea. This motor protein contributes to outer hair cell motility. At present, it is not clear how the interference of prestin function affects cochlear responses to acoustic overstimulation. To address this question, a genetic model of prestin dysfunction in mice was created by inserting an internal ribosome entry site (IRES)-CreER^T2-FRT-Neo-FRT cassette into the prestin locus after the stop codon. Homozygous mice exhibit a threshold elevation of auditory brainstem responses with large individual variation. These mice also display a threshold elevation and a shift of the input/output function of the distortion product otoacoustic emission, suggesting a reduction in outer hair cell function. The disruption of prestin function reduces the threshold shifts caused by exposure to a loud noise at 120 dB (sound pressure level) for 1 h. This reduction is positively correlated with the level of pre-noise cochlear dysfunction and is accompanied by a reduced change in Cdh1 expression, suggesting a reduction in molecular responses to the acoustic overstimulation. Together, these results suggest that prestin interference reduces cochlear stress responses to acoustic overstimulation.     

Regeneration of Stereocilia of Hair Cells by Forced Atoh1 Expression in the Adult Mammalian Cochlea

The hallmark of mechanosensory hair cells is the stereocilia, where mechanical stimuli are converted into electrical signals. These delicate stereocilia are susceptible to acoustic trauma and ototoxic drugs. While hair cells in lower vertebrates and the mammalian vestibular system can spontaneously regenerate lost stereocilia, mammalian cochlear hair cells no longer retain this capability. We explored the possibility of regenerating stereocilia in the noise-deafened guinea pig cochlea by cochlear inoculation of a viral vector carrying Atoh1, a gene critical for hair cell differentiation. Exposure to simulated gunfire resulted in a 60–70 dB hearing loss and extensive damage and loss of stereocilia bundles of both inner and outer hair cells along the entire cochlear length. However, most injured hair cells remained in the organ of Corti for up to 10 days after the trauma. A viral vector carrying an EGFP-labeled Atoh1 gene was inoculated into the cochlea through the round window on the seventh day after noise exposure. Auditory brainstem response measured one month after inoculation showed that hearing thresholds were substantially improved. Scanning electron microscopy revealed that the damaged/lost stereocilia bundles were repaired or regenerated after Atoh1 treatment, suggesting that Atoh1 was able to induce repair/regeneration of the damaged or lost stereocilia. Therefore, our studies revealed a new role of Atoh1 as a gene critical for promoting repair/regeneration of stereocilia and maintaining injured hair cells in the adult mammal cochlea. Atoh1-based gene therapy, therefore, has the potential to treat noise-induced hearing loss if the treatment is carried out before hair cells die.     

Removal of immunoglobulin-like domains from titin’s spring segment alters titin splicing in mouse skeletal muscle and causes myopathy

Titin is a molecular spring that determines the passive stiffness of muscle cells. Changes in titin’s stiffness occur in various myopathies, but whether these are a cause or an effect of the disease is unknown. We studied a novel mouse model in which titin’s stiffness was slightly increased by deleting nine immunoglobulin (Ig)-like domains from titin’s constitutively expressed proximal tandem Ig segment (IG KO). KO mice displayed mild kyphosis, a phenotype commonly associated with skeletal muscle myopathy. Slow muscles were atrophic with alterations in myosin isoform expression; functional studies in soleus muscle revealed a reduced specific twitch force. Exon expression analysis showed that KO mice underwent additional changes in titin splicing to yield smaller than expected titin isoforms that were much stiffer than expected. Additionally, splicing occurred in the PEVK region of titin, a finding confirmed at the protein level. The titin-binding protein Ankrd1 was highly increased in the IG KO, but this did not play a role in generating small titin isoforms because titin expression was unaltered in IG KO mice crossed with Ankrd1-deficient mice. In contrast, the splicing factor RBM20 (RNA-binding motif 20) was also significantly increased in IG KO mice, and additional differential splicing was reversed in IG KO mice crossed with a mouse with reduced RBM20 activity. Thus, increasing titin’s stiffness triggers pathological changes in skeletal muscle, with an important role played by RBM20.     

The Muscle Ankyrin Repeat Proteins CARP,Ankrd2, and DARP Are Not Essential for Normal Cardiac Development and Function at Basal Conditions and in Response to Pressure Overload

Ankrd1/CARP, Ankrd2/Arpp, and Ankrd23/DARP belong to a family of stress inducible ankyrin repeat proteins expressed in striated muscle (MARPs). The MARPs are homologous in structure and localized in the nucleus where they negatively regulate gene expression as well as in the sarcomeric I-band, where they are thought to be involved in mechanosensing. Together with their strong induction during cardiac disease and the identification of causative Ankrd1 gene mutations in cardiomyopathy patients, this suggests their important roles in cardiac development, function, and disease. To determine the functional role of MARPs in vivo, we studied knockout (KO) mice of each of the three family members. Single KO mice were viable and had no apparent cardiac phenotype. We therefore hypothesized that the three highly homologous MARP proteins may have redundant functions in the heart and studied double and triple MARP KO mice. Unexpectedly, MARP triple KO mice were viable and had normal cardiac function both at basal levels and in response to mechanical pressure overload induced by transverse aortic constriction as assessed by echocardiography and hemodynamic studies. Thus, CARP, Ankrd2, and DARP are not essential for normal cardiac development and function at basal conditions and in response to mechanical pressure overload.     

The novel mouse mutation Oblivion inactivates the PMCA2 pump and causes progressive hearing loss

Progressive hearing loss is common in the human population, but we have few clues to the molecular basis. Mouse mutants with progressive hearing loss offer valuable insights, and ENU (N-ethyl-N-nitrosourea) mutagenesis is a useful way of generating models. We have characterised a new ENU-induced mouse mutant, Oblivion (allele symbol Obl), showing semi-dominant inheritance of hearing impairment. Obl/+ mutants showed increasing hearing impairment from post-natal day (P)20 to P90, and loss of auditory function was followed by a corresponding base to apex progression of hair cell degeneration. Obl/Obl mutants were small, showed severe vestibular dysfunction by 2 weeks of age, and were completely deaf from birth; sensory hair cells were completely degenerate in the basal turn of the cochlea, although hair cells appeared normal in the apex. We mapped the mutation to Chromosome 6. Mutation analysis of Atp2b2 showed a missense mutation (2630C→T) in exon 15, causing a serine to phenylalanine substitution (S877F) in transmembrane domain 6 of the PMCA2 pump, the resident Ca²⁺ pump of hair cell stereocilia. Transmembrane domain mutations in these pumps generally are believed to be incompatible with normal targeting of the protein to the plasma membrane. However, analyses of hair cells in cultured utricular maculae of Obl/Obl mice and of the mutant Obl pump in model cells showed that the protein was correctly targeted to the plasma membrane. Biochemical and biophysical characterisation showed that the pump had lost a significant portion of its non-stimulated Ca²⁺ exporting ability. These findings can explain the progressive loss of auditory function, and indicate the limits in our ability to predict mechanism from sequence alone.     

A mutation in synaptojanin 2 causes progressive hearing loss in the ENU-mutagenised mouse strain Mozart

Background

Hearing impairment is the most common sensory impairment in humans, affecting 1∶1,000 births. We have identified an ENU generated mouse mutant, Mozart, with recessively inherited, non-syndromic progressive hearing loss caused by a mutation in the synaptojanin 2 (Synj2), a central regulatory enzyme in the phosphoinositide-signaling cascade.

Methodology/Principal Findings

The hearing loss in Mozart is caused by a p.Asn538Lys mutation in the catalytic domain of the inositol polyphosphate 5-phosphatase synaptojanin 2. Within the cochlea, Synj2 mRNA expression was detected in the inner and outer hair cells but not in the spiral ganglion. Synj2 ^N538K mutant protein showed loss of lipid phosphatase activity, and was unable to degrade phosphoinositide signaling molecules. Mutant Mozart mice (Synj2 ^N538K/N538K) exhibited progressive hearing loss and showed signs of hair cell degeneration as early as two weeks of age, with fusion of stereocilia followed by complete loss of hair bundles and ultimately loss of hair cells. No changes in vestibular or neurological function, or other clinical or behavioral manifestations were apparent.

Conclusions/Significance

Phosphoinositides are membrane associated signaling molecules that regulate many cellular processes including cell death, proliferation, actin polymerization and ion channel activity. These results reveal Synj2 as a critical regulator of hair cell survival that is essential for hair cell maintenance and hearing function.     

Prestin-Dependence of Outer Hair Cell Survival and Partial Rescue of Outer Hair Cell Loss in Prestin V499G/Y501H Knockin Mice

A knockin (KI) mouse expressing mutated prestin ^V499G/Y501H (499 prestin) was created to study cochlear amplification. Recordings from isolated outer hair cells (OHC) in this mutant showed vastly reduced electromotility and, as a consequence, reduced hearing sensitivity. Although 499 prestin OHCs were normal in stiffness and longer than OHCs lacking prestin, accelerated OHC death was unexpectedly observed relative to that documented in prestin knockout (KO) mice. These observations imply an additional role of prestin in OHC maintenance besides its known requirement for mammalian cochlear amplification. In order to gain mechanistic insights into prestin-associated OHC loss, we implemented several interventions to improve survival. First, 499 prestin KI’s were backcrossed to Bak KO mice, which lack the mitochondrial pro-apoptotic gene Bak. Because oxidative stress is implicated in OHC death, another group of 499 prestin KI mice was fed the antioxidant diet, Protandim. 499 KI mice were also backcrossed onto the FVB murine strain, which retains excellent high-frequency hearing well into adulthood, to reduce the compounding effect of age-related hearing loss associated with the original 499 prestin KIs. Finally, a compound heterozygous (chet) mouse expressing one copy of 499 prestin and one copy of KO prestin was also created to reduce quantities of 499 prestin protein. Results show reduction in OHC death in chets, and in 499 prestin KIs on the FVB background, but only a slight improvement in OHC survival for mice receiving Protandim. We also report that improved OHC survival in 499 prestin KIs had little effect on hearing phenotype, reaffirming the original contention about the essential role of prestin’s motor function in cochlear amplification.     

Mutations in Grxcr1 Are The Basis for Inner Ear Dysfunction in the Pirouette Mouse

Recessive mutations at the mouse pirouette (pi) locus result in hearing loss and vestibular dysfunction due to neuroepithelial defects in the inner ear. Using a positional cloning strategy, we have identified mutations in the gene Grxcr1 (glutaredoxin cysteine-rich 1) in five independent allelic strains of pirouette mice. We also provide sequence data of GRXCR1 from humans with profound hearing loss suggesting that pirouette is a model for studying the mechanism of nonsyndromic deafness DFNB25. Grxcr1 encodes a 290 amino acid protein that contains a region of similarity to glutaredoxin proteins and a cysteine-rich region at its C terminus. Grxcr1 is expressed in sensory epithelia of the inner ear, and its encoded protein is localized along the length of stereocilia, the actin-filament-rich mechanosensory structures at the apical surface of auditory and vestibular hair cells. The precise architecture of hair cell stereocilia is essential for normal hearing. Loss of function of Grxcr1 in homozygous pirouette mice results in abnormally thin and slightly shortened stereocilia. When overexpressed in transfected cells, GRXCR1 localizes along the length of actin-filament-rich structures at the dorsal-apical surface and induces structures with greater actin filament content and/or increased lengths in a subset of cells. Our results suggest that deafness in pirouette mutants is associated with loss of GRXCR1 function in modulating actin cytoskeletal architecture in the developing stereocilia of sensory hair cells.     

Mutations in LOXHD1, an Evolutionarily Conserved Stereociliary Protein, Disrupt Hair Cell Function in Mice and Cause Progressive Hearing Loss in Humans

Hearing loss is the most common form of sensory impairment in humans and is frequently progressive in nature. Here we link a previously uncharacterized gene to hearing impairment in mice and humans. We show that hearing loss in the ethylnitrosourea (ENU)-induced samba mouse line is caused by a mutation in Loxhd1. LOXHD1 consists entirely of PLAT (polycystin/lipoxygenase/α-toxin) domains and is expressed along the membrane of mature hair cell stereocilia. Stereociliary development is unaffected in samba mice, but hair cell function is perturbed and hair cells eventually degenerate. Based on the studies in mice, we screened DNA from human families segregating deafness and identified a mutation in LOXHD1, which causes DFNB77, a progressive form of autosomal-recessive nonsyndromic hearing loss (ARNSHL). LOXHD1, MYO3a, and PJVK are the only human genes to date linked to progressive ARNSHL. These three genes are required for hair cell function, suggesting that age-dependent hair cell failure is a common mechanism for progressive ARNSHL.     

A mouse model for human hearing loss DFNB30 due to loss of function of myosin IIIA

The motor protein myosin IIIA is critical for maintenance of normal hearing. Homozygosity and compound heterozygosity for loss-of-function mutations in MYO3A, which encodes myosin IIIA, are responsible for inherited human progressive hearing loss DFNB30. To further evaluate this hearing loss, we constructed a mouse model, Myo3a ^KI/KI , that harbors the mutation equivalent to the nonsense allele responsible for the most severe human phenotype. Myo3a ^KI/KI mice were compared to their wild-type littermates. Myosin IIIA, with a unique N-terminal kinase domain and a C-terminal actin-binding domain, localizes to the tips of stereocilia in wild-type mice but is absent in the mutant. The phenotype of the Myo3a ^KI/KI mouse parallels the phenotype of human DFNB30. Hearing loss, as measured by auditory brainstem response, is reduced and progresses significantly with age. Vestibular function is normal. Outer hair cells of Myo3a ^KI/KI mice degenerate with age in a pattern consistent with their progressive hearing loss.     

METTL14 is essential for β-cell survival and insulin secretion

Defects in the development, maintenance or expansion of β-cell mass can result in impaired glucose metabolism and diabetes. N⁶-methyladenosine affects mRNA stability and translation efficiency, and impacts cell differentiation and stress response. To determine if there is a role for m⁶A in β-cells, we investigated the effect of Mettl14, a key component of the m⁶A methyltransferase complex, on β-cell survival and function using rat insulin-2 promoter-Cre-mediated deletion of Mettl14 mouse line (βKO). We found that βKO mice with normal chow exhibited glucose intolerance, lower levels of glucose-stimulated insulin secretion, increased β-cell death and decreased β-cell mass. In addition, HFD-fed βKO mice developed glucose intolerance, decreased β-cell mass and proliferation, exhibited lower body weight, increased adipose tissue mass, and enhanced insulin sensitivity due to enhanced AKT signaling and decreased gluconeogenesis in the liver. HFD-fed βKO mice also showed a decrease in de novo lipogenesis, and an increase in lipolysis in the liver. RNA sequencing in islets revealed that Mettl14 deficiency in β-cells altered mRNA expression levels of some genes related to cell death and inflammation. Together, we showed that Mettl14 in β-cells plays a key role in β-cell survival, insulin secretion and glucose homeostasis.