A spontaneous mouse deletion in Mctp1 uncovers a long-range cis-regulatory region crucial for NR2F1 function during inner ear development.

Hundreds of thousands of cis-regulatory DNA sequences are predicted in vertebrate genomes, but unlike genes themselves, few have been characterized at the functional level or even unambiguously paired with a target gene. Here we serendipitously identified and started investigating the first reported long-range regulatory region for the Nr2f1 (Coup-TFI) transcription factor gene. NR2F1 is temporally and spatially regulated during development and required for patterning and regionalization in the nervous system, including sensory hair cell organization in the auditory epithelium of the cochlea. Analyzing the deaf wanderer (dwnd) spontaneous mouse mutation, we traced back the cause of its associated circling behavior to a 53 kb deletion removing five exons and adjacent intronic regions of the poorly characterized Mctp1 gene. Interestingly, loss of Mctp1 function cannot account for the hearing loss, inner ear dysmorphology and sensory hair cell disorganization observed in dwnd mutants. Instead, we found that the Mctp1dwnd deletion affects the Nr2f1 gene located 1.4 Mb away, downregulating transcription and protein expression in the embryonic cochlea. Remarkably, the Mctp1dwnd allele failed to complement a targeted inactivation allele of Nr2f1, and transheterozygotes or Mctp1dwnd homozygotes exhibit the same morphological defects observed in inner ears of Nr2f1 mutants without sharing their early life lethality. Defects include improper separation of the utricle and saccule in the vestibule not described previously, which can explain the circling behavior that first brought the spontaneous mutation to attention. By contrast, mice homozygous for a targeted inactivation of Mctp1 have normal hearing and inner ear structures. We conclude that the 53 kb Mctp1dwnd deletion encompasses a long-range cis-regulatory region essential for proper Nr2f1 expression in the embryonic inner ear, providing a first opportunity to investigate Nr2f1 function in postnatal inner ears. This work adds to the short list of long-range regulatory regions characterized as essential to drive expression of key developmental control genes.

Deletion of a Long-Range Dlx5 Enhancer Disrupts Inner Ear Development in Mice.

Distal enhancers are thought to play important roles in the spatiotemporal regulation of gene expression during embryonic development, but few predicted enhancer elements have been shown to affect transcription of their endogenous genes or to alter phenotypes when disrupted. Here, we demonstrate that a 123.6-kb deletion within the mouse Slc25a13 gene is associated with reduced transcription of Dlx5, a gene located 660 kb away. Mice homozygous for the Slc25a13 deletion mutation [named hyperspin (hspn)] have malformed inner ears and are deaf with balance defects, whereas previously reported Slc25a13 knockout mice showed no phenotypic abnormalities. Inner ears of Slc25a13hspn/hspn mice have malformations similar to those of Dlx5-/- embryos, and Dlx5 expression is severely reduced in the otocyst but not the branchial arches of Slc25a13hspn/hspn embryos, indicating that the Slc25a13hspn deletion affects otic-specific enhancers of Dlx5 In addition, transheterozygous Slc25a13+/hspn Dlx5+/- mice exhibit noncomplementation with inner ear dysmorphologies similar to those of Slc25a13hspn/hspn and Dlx5-/-embryos, verifying a cis-acting effect of the Slc25a13hspn deletion on Dlx5 expression. CRISPR/Cas9-mediated deletions of putative enhancer elements located within the Slc25a13hspn deleted region failed to phenocopy the defects of Slc25a13hspn/hspn mice, suggesting the possibility of multiple enhancers with redundant functions. Our findings in mice suggest that analogous enhancer elements in the human SLC25A13 gene may regulate DLX5 expression and underlie the hearing loss that is associated with split-hand/-foot malformation 1 syndrome. Slc25a13hspn/hspn mice provide a new animal model for studying long-range enhancer effects on Dlx5 expression in the developing inner ear.

Hearing loss without overt metabolic acidosis in ATP6V1B1 deficient MRL mice, a new genetic model for non-syndromic deafness with enlarged vestibular aqueducts.

Mutations of the human ATP6V1B1 gene cause distal renal tubular acidosis (dRTA; OMIM #267300) often associated with sensorineural hearing impairment; however, mice with a knockout mutation of Atp6v1b1 were reported to exhibit a compensated acidosis and normal hearing. We discovered a new spontaneous mutation (vortex, symbol vtx) of Atp6v1b1 in an MRL/MpJ (MRL) colony of mice. In contrast to the reported phenotype of the knockout mouse, which was developed on a primarily C57BL/6 (B6) strain background, MRL-Atp6v1b1vtx/vtx mutant mice exhibit profound hearing impairment, which is associated with enlarged endolymphatic compartments of the inner ear. Mutant mice have alkaline urine but do not exhibit overt metabolic acidosis, a renal phenotype similar to that of the Atpbv1b1 knockout mouse. The abnormal inner ear phenotype of MRL- Atp6v1b1vtx/vtx mice was lost when the mutation was transferred onto the C57BL/6J (B6) background, indicating the influence of strain-specific genetic modifiers. To genetically map modifier loci in Atp6v1b1vtx/vtx mice, we analysed ABR thresholds of progeny from a backcross segregating MRL and B6 alleles. We found statistically significant linkage with a locus on Chr 13 that accounts for about 20% of the hearing threshold variation in the backcross mice. The important effect that genetic background has on the inner ear phenotype of Atp6v1b1 mutant mice provides insight into the hearing loss variability associated with dRTA caused by ATP6V1B1 mutations. Because MRL-Atp6v1b1vxt/vtx mice do not recapitulate the metabolic acidosis of dRTA patients, they provide a new genetic model for nonsyndromic deafness with enlarged vestibular aqueduct (EVA; OMIM #600791).

N-acetyl-cysteine prevents age-related hearing loss and the progressive loss of inner hair cells in γ-glutamyl transferase 1 deficient mice.

Genetic factors combined with oxidative stress are major determinants of age-related hearing loss (ARHL), one of the most prevalent disorders of the elderly. Dwarf grey mice, Ggt1dwg/dwg, are homozygous for a loss of function mutation of the g-glutamyl transferase 1 gene, which encodes an important antioxidant enzyme critical for the resynthesis of glutathione (GSH). Since GSH reduces oxidative damage, we hypothesized that Ggt1dwg/dwg mice would be susceptible to ARHL. Surprisingly, otoacoustic emissions and cochlear microphonic potentials, which reflect cochlear outer hair cell (OHC) function, were largely unaffected in mutant mice, whereas auditory brainstem responses and the compound action potential were grossly abnormal. These functional deficits were associated with an unusual and selective loss of inner hair cells (IHC), but retention of OHC and auditory nerve fibers. Remarkably, hearing deficits and IHC loss were completely prevented by N-acetyl-L-cysteine, which induces de novo synthesis of GSH; however, hearing deficits and IHC loss reappeared when treatment was discontinued. Ggt1dwg/dwg mice represent an important new model for investigating ARHL, therapeutic interventions, and understanding the perceptual and electrophysiological consequences of sensory deprivation caused by the loss of sensory input exclusively from IHC.

A QTL on Chr 5 modifies hearing loss associated with the fascin-2 variant of DBA/2J mice.

Inbred mouse strains serve as important models for human presbycusis or age-related hearing loss. We previously mapped a locus (ahl8) contributing to the progressive hearing loss of DBA/2J (D2) mice and later showed that a missense variant of the Fscn2 gene, unique to the D2 inbred strain, was responsible for the ahl8 effect. Although ahl8 can explain much of the hearing loss difference between C57BL/6J (B6) and D2 strain mice, other loci also contribute. Here, we present results of our linkage analyses to map quantitative trait loci (QTLs) that modify the severity of hearing loss associated with the D2 strain Fscn2 (ahl8) allele. We searched for modifier loci by analyzing 31 BXD recombinant inbred (RI) lines fixed for the predisposing D2-derived Fscn2 (ahl8/ahl8) genotype and found a statistically significant linkage association of threshold means with a QTL on Chr 5, which we designated M5ahl8. The highest association (LOD 4.6) was with markers at the 84-90 Mb position of Chr 5, which could explain about 46 % of the among-RI strain variation in auditory brainstem response (ABR) threshold means. The semidominant nature of the modifying effect of M5ahl8 on the Fscn2 (ahl8/ahl8) phenotype was demonstrated by analysis of a backcross involving D2 and B6.D2-Chr11D/LusJ strain mice. The Chr 5 map position of M5ahl8 and the D2 origin of its susceptibility allele correspond to Tmc1m4, a previously reported QTL that modifies outer hair cell degeneration in Tmc1 (Bth) mutant mice, suggesting that M5ahl8 and Tmc1m4 may represent the same gene affecting maintenance of stereocilia structure and function during aging.

Hearing impairment in hypothyroid dwarf mice caused by mutations of the thyroid peroxidase gene.

Thyroid hormone (TH) is essential for proper cochlear development and function, and TH deficiencies cause variable hearing impairment in humans and mice. Thyroid peroxidase (TPO) catalyzes key reactions in TH synthesis, and TPO mutations have been found to underlie many cases of congenital hypothyroidism in human patients. In contrast, only a single mutation of the mouse TPO gene has been reported previously (Tpo(R479C)) but was not evaluated for auditory function. Here, we describe and characterize two new mouse mutations of Tpo with an emphasis on their associated auditory deficits. Mice homozygous for these recessive mutations have dysplastic thyroid glands and lack detectable levels of TH. Because of the small size of mutant mice, the mutations were named teeny (symbol Tpo(tee)) and teeny-2 Jackson (Tpo(tee-2J)). Tpo(tee) is a single base-pair missense mutation that was induced by ENU, and Tpo(tee-2J) is a 64 bp intragenic deletion that arose spontaneously. The Tpo(tee) mutation changes the codon for a highly conserved tyrosine to asparagine (p.Y614N), and the Tpo(tee-2J) mutation deletes a splice donor site, which results in exon skipping and aberrant transcripts. Mutant mice are profoundly hearing impaired with auditory brainstem response (ABR) thresholds about 60 dB above those of non-mutant controls. The maturation of cochlear structures is delayed in mutant mice and tectorial membranes are abnormally thick. To evaluate the effect of genetic background on auditory phenotype, we produced a C3.B6-Tpo(tee-2J) congenic strain and found that ABR thresholds of mutant mice on the C3H/HeJ strain background are 10-12 dB lower than those of mutant mice on the C57BL/6 J background. The Tpo mutant strains described here provide new heritable mouse models of congenital hypothyroidism that will be valuable for future studies of thyroid hormones' role in auditory development and function.

Mutations of the mouse ELMO domain containing 1 gene (Elmod1) link small GTPase signaling to actin cytoskeleton dynamics in hair cell stereocilia.

Stereocilia, the modified microvilli projecting from the apical surfaces of the sensory hair cells of the inner ear, are essential to the mechanoelectrical transduction process underlying hearing and balance. The actin-filled stereocilia on each hair cell are tethered together by fibrous links to form a highly patterned hair bundle. Although many structural components of hair bundles have been identified, little is known about the signaling mechanisms that regulate their development, morphology, and maintenance. Here, we describe two naturally occurring, allelic mutations that result in hearing and balance deficits in mice, named roundabout (rda) and roundabout-2J (rda(2J)). Positional cloning identified both as mutations of the mouse ELMO domain containing 1 gene (Elmod1), a poorly characterized gene with no previously reported mutant phenotypes. The rda mutation is a 138 kb deletion that includes exons 1-5 of Elmod1, and rda(2J) is an intragenic duplication of exons 3-8 of Elmod1. The deafness associated with these mutations is caused by cochlear hair cell dysfunction, as indicated by conspicuous elongations and fusions of inner hair cell stereocilia and progressive degeneration of outer hair cell stereocilia. Mammalian ELMO-family proteins are known to be involved in complexes that activate small GTPases to regulate the actin cytoskeleton during phagocytosis and cell migration. ELMOD1 and ELMOD2 recently were shown to function as GTPase-activating proteins (GAPs) for the Arf family of small G proteins. Our finding connecting ELMOD1 deficiencies with stereocilia dysmorphologies thus establishes a link between the Ras superfamily of small regulatory GTPases and the actin cytoskeleton dynamics of hair cell stereocilia.

Association of a citrate synthase missense mutation with age-related hearing loss in A/J mice.

We previously mapped a locus (ahl4) on distal Chromosome 10 that contributes to the age-related hearing loss of A/J strain mice. Here, we report on a refined genetic map position for ahl4 and its association with a mutation in the citrate synthase gene (Cs). We mapped ahl4 to the distal-most 7 megabases (Mb) of chromosome 10 by analysis of a new linkage backcross and then further narrowed the interval to 5.5 Mb by analysis of 8 C57BL/6J congenic lines with different A/J-derived segments of chromosome 10. A nucleotide variant in exon 3 of Cs is the only known DNA difference within the ahl4 candidate gene interval that is unique to the A/J strain and that causes a nonsynonymous codon change. Multiple lines of evidence implicate this missense mutation (H55N) as the underlying cause of ahl4-related hearing loss, likely through its effects on mitochondrial adenosine trisphosphate (ATP) and free radical production in cochlear hair cells. The A/J mouse thus provides a new model system for in vivo studies of mitochondrial function and hearing loss.

Genetic background effects on age-related hearing loss associated with Cdh23 variants in mice.

Inbred strain variants of the Cdh23 gene have been shown to influence the onset and progression of age-related hearing loss (AHL) in mice. In linkage backcrosses, the recessive Cdh23 allele (ahl) of the C57BL/6J strain, when homozygous, confers increased susceptibility to AHL, while the dominant allele (Ahl+) of the CBA/CaJ strain confers resistance. To determine the isolated effects of these alleles on different strain backgrounds, we produced the reciprocal congenic strains B6.CBACa-Cdh23(Ahl)(+) and CBACa.B6-Cdh23(ahl) and tested 15-30 mice from each for hearing loss progression. ABR thresholds for 8 kHz, 16 kHz, and 32 kHz pure-tone stimuli were measured at 3, 6, 9, 12, 15 and 18 months of age and compared with age-matched mice of the C57BL/6J and CBA/CaJ parental strains. Mice of the C57BL/6N strain, which is the source of embryonic stem cells for the large International Knockout Mouse Consortium, were also tested for comparisons with C57BL/6J mice. Mice of the C57BL/6J and C57BL/6N strains exhibited identical hearing loss profiles: their 32 kHz ABR thresholds were significantly higher than those of CBA/CaJ and congenic strain mice by 6 months of age, and their 16 kHz thresholds were significantly higher by 12 months. Thresholds of the CBA/CaJ, the B6.CBACa-Cdh23(Ahl)(+), and the CBACa.B6-Cdh23(ahl) strain mice differed little from one another and only slightly increased throughout the 18-month test period. Hearing loss, which corresponded well with cochlear hair cell loss, was most profound in the C57BL/6J and C57BL/6NJ strains. These results indicate that the CBA/CaJ-derived Cdh23(Ahl)(+) allele dramatically lessens hearing loss and hair cell death in an otherwise C57BL/6J genetic background, but that the C57BL/6J-derived Cdh23(ahl) allele has little effect on hearing loss in an otherwise CBA/CaJ background. We conclude that although Cdh23(ahl) homozygosity is necessary, it is not by itself sufficient to account for the accelerated hearing loss of C57BL/6J mice.

A modifier gene alleviates hypothyroidism-induced hearing impairment in Pou1f1dw dwarf mice.

Thyroid hormone has pleiotropic effects on cochlear development, and genomic variation influences the severity of associated hearing deficits. DW/J-Pou1f1dw/dw mutant mice lack pituitary thyrotropin, which causes severe thyroid hormone deficiency and profound hearing impairment. To assess the genetic complexity of protective effects on hypothyroidism-induced hearing impairment, an F1 intercross was generated between DW/J-Pou1f1dw/+ carriers and an inbred strain with excellent hearing derived from Mus castaneus, CAST/EiJ. Approximately 24% of the (DW/J×CAST/EiJ) Pou1f1dw/dw F2 progeny had normal hearing. A genome scan revealed a locus on chromosome 2, named modifier of dw hearing, or Mdwh, that rescues hearing despite persistent hypothyroidism. This chromosomal region contains the modifier of tubby hearing 1 (Moth1) locus that encodes a protective allele of the microtubule-associated protein MTAP1A. DW/J-Pou1f1dw/+ carriers were crossed with the AKR strain, which also carries a protective allele of Mtap1a, and we found that AKR is not protective for hearing in the (DW/J×AKR) Pou1f1dw/dw F2 progeny. Thus, protective alleles of Mtap1a are not sufficient to rescue DW/J-Pou1f1dw/dw hearing. We expect that identification of protective modifiers will enhance our understanding of the mechanisms of hypothyroidism-induced hearing impairment.

Alström Syndrome protein ALMS1 localizes to basal bodies of cochlear hair cells and regulates cilium-dependent planar cell polarity.

Alström Syndrome is a life-threatening disease characterized primarily by numerous metabolic abnormalities, retinal degeneration, cardiomyopathy, kidney and liver disease, and sensorineural hearing loss. The cellular localization of the affected protein, ALMS1, has suggested roles in ciliary function and/or ciliogenesis. We have investigated the role of ALMS1 in the cochlea and the pathogenesis of hearing loss in Alström Syndrome. In neonatal rat organ of Corti, ALMS1 was localized to the basal bodies of hair cells and supporting cells. ALMS1 was also evident at the basal bodies of differentiating fibrocytes and marginal cells in the lateral wall. Centriolar ALMS1 expression was retained into maturity. In Alms1-disrupted mice, which recapitulate the neurosensory deficits of human Alström Syndrome, cochleae displayed several cyto-architectural defects including abnormalities in the shape and orientation of hair cell stereociliary bundles. Developing hair cells were ciliated, suggesting that ciliogenesis was largely normal. In adult mice, in addition to bundle abnormalities, there was an accelerated loss of outer hair cells and the progressive appearance of large lesions in stria vascularis. Although the mice progressively lost distortion product otoacoustic emissions, suggesting defects in outer hair cell amplification, their endocochlear potentials were normal, indicating the strial atrophy did not affect its function. These results identify previously unrecognized cochlear histopathologies associated with this ciliopathy that (i) implicate ALMS1 in planar cell polarity signaling and (ii) suggest that the loss of outer hair cells causes the majority of the hearing loss in Alström Syndrome.

NPHP4 is necessary for normal photoreceptor ribbon synapse maintenance and outer segment formation, and for sperm development.

Nephronophthisis (NPHP) is an autosomal recessive kidney disease that is often associated with vision and/or brain defects. To date, 11 genes are known to cause NPHP. The gene products, while structurally unrelated, all localize to cilia or centrosomes. Although mouse models of NPHP are available for 9 of the 11 genes, none has been described for nephronophthisis 4 (Nphp4). Here we report a novel, chemically induced mutant, nmf192, that bears a nonsense mutation in exon 4 of Nphp4. Homozygous mutant Nphp4(nmf192/nmf192) mice do not exhibit renal defects, phenotypes observed in human patients bearing mutations in NPHP4, but they do develop severe photoreceptor degeneration and extinguished rod and cone ERG responses by 9 weeks of age. Photoreceptor outer segments (OS) fail to develop properly, and some OS markers mislocalize to the inner segments and outer nuclear layer in the Nphp4(nmf192/nmf192) mutant retina. Despite NPHP4 localization to the transition zone in the connecting cilia (CC), the CC appear to be normal in structure and ciliary transport function is partially retained. Likewise, synaptic ribbons develop normally but then rapidly degenerate by P14. Finally, Nphp4(nmf192/nmf192) male mutants are sterile and show reduced sperm motility and epididymal sperm counts. Although Nphp4(nmf192/nmf192) mice fail to recapitulate the kidney phenotype of NPHP, they will provide a valuable tool to further elucidate how NPHP4 functions in the retina and male reproductive organs.

The R109H variant of fascin-2, a developmentally regulated actin crosslinker in hair-cell stereocilia, underlies early-onset hearing loss of DBA/2J mice.

The quantitative trait locus ahl8 is a key contributor to the early-onset, age-related hearing loss of DBA/2J mice. A nonsynonymous nucleotide substitution in the mouse fascin-2 gene (Fscn2) is responsible for this phenotype, confirmed by wild-type BAC transgene rescue of hearing loss in DBA/2J mice. In chickens and mice, FSCN2 protein is abundant in hair-cell stereocilia, the actin-rich structures comprising the mechanically sensitive hair bundle, and is concentrated toward stereocilia tips of the bundle's longest stereocilia. FSCN2 expression increases when these stereocilia differentially elongate, suggesting that FSCN2 controls filament growth, stiffens exposed stereocilia, or both. Because ahl8 accelerates hearing loss only in the presence of mutant cadherin 23, a component of hair-cell tip links, mechanotransduction and actin crosslinking must be functionally interrelated.

Ush1c gene expression levels in the ear and eye suggest different roles for Ush1c in neurosensory organs in a new Ush1c knockout mouse.

Usher syndrome (USH) is the most common form of deaf-blindness in humans. Molecular characterization revealed that the USH gene products form a macromolecular protein network in hair cells of the inner ear and in photoreceptor cells of the retina via binding to PDZ domains in the scaffold protein harmonin encoded by the Ush1c gene in mice and humans. Although several mouse mutants for the Ush1c gene have been described, we generated a targeted null mutation Ush1c mouse model in which the first four exons of the Ush1c gene were replaced with a reporter gene. Here, we assessed the expression pattern of the reporter gene under control of Ush1c regulatory elements and characterized the phenotype of mice defective for Ush1c. These Ush1 knockout mice are deaf but do not recapitulate vision defects before 10 months of age. Our data show LacZ expression in multiple layers of the retina but in neither outer nor inner segments of the photoreceptor layers in mice bearing the knockout construct at 1-5 months of age. The fact that Ush1c expression is much higher in the ear than in the eye suggests a different role for Ush1c in ear function than in the eye and may explain why Ush1c mutant mice do not recapitulate vision defects.

A locus on distal chromosome 11 (ahl8) and its interaction with Cdh23 ahl underlie the early onset, age-related hearing loss of DBA/2J mice.

The DBA/2J inbred strain of mice is used extensively in hearing research, yet little is known about the genetic basis for its early onset, progressive hearing loss. To map underlying genetic factors we analyzed recombinant inbred strains and linkage backcrosses. Analysis of 213 mice from 31 BXD recombinant inbred strains detected linkage of auditory brain-stem response thresholds with a locus on distal chromosome 11, which we designate ahl8. Analysis of 225 N2 mice from a backcross of (C57BL/6JxDBA/2J) F1 hybrids to DBA/2J mice confirmed this linkage (LOD>50) and refined the ahl8 candidate gene interval. Analysis of 214 mice from a backcross of (B6.CAST-Cdh23 Ahl+ xDBA/2J) F1 hybrids to DBA/2J mice demonstrated a genetic interaction of Cdh23 with ahl8. We conclude that ahl8 is a major contributor to the hearing loss of DBA/2J mice and that its effects are dependent on the predisposing Cdh23 ahl genotype of this strain.

Mutation of the Cyba gene encoding p22phox causes vestibular and immune defects in mice.

In humans, hereditary inactivation of either p22(phox) or gp91(phox) leads to chronic granulomatous disease (CGD), a severe immune disorder characterized by the inability of phagocytes to produce bacteria-destroying ROS. Heterodimers of p22(phox) and gp91(phox) proteins constitute the superoxide-producing cytochrome core of the phagocyte NADPH oxidase. In this study, we identified the nmf333 mouse strain as what we believe to be the first animal model of p22(phox) deficiency. Characterization of nmf333 mice revealed that deletion of p22(phox) inactivated not only the phagocyte NADPH oxidase, but also a second cytochrome in the inner ear epithelium. As a consequence, mice of the nmf333 strain exhibit a compound phenotype consisting of both a CGD-like immune defect and a balance disorder caused by the aberrant development of gravity-sensing organs. Thus, in addition to identifying a model of p22(phox)-dependent immune deficiency, our study indicates that a clinically identifiable patient population with an otherwise cryptic loss of gravity-sensor function may exist. Thus, p22(phox) represents a shared and essential component of at least 2 superoxide-producing cytochromes with entirely different biological functions. The site of p22(phox) expression in the inner ear leads us to propose what we believe to be a novel mechanism for the control of vestibular organogenesis.

A missense mutation in the conserved C2B domain of otoferlin causes deafness in a new mouse model of DFNB9.

Mutations of the otoferlin gene have been shown to underlie deafness disorders in humans and mice. Analyses of genetically engineered mice lacking otoferlin have demonstrated an essential role for this protein in vesicle exocytosis at the inner hair cell afferent synapse. Here, we report on the molecular and phenotypic characterization of a new ENU-induced missense mutation of the mouse otoferlin gene designated Otof(deaf5Jcs). The mutation is a single T to A base substitution in exon 10 of Otof that causes a non-conservative amino acid change of isoleucine to asparagine in the C2B domain of the protein. Although strong immunoreactivity with an otoferlin-specific antibody was detected in cochlear hair cells of wildtype mice, no expression was detected in mutant mice, indicating that the missense mutation has a severe effect on the stability of the protein and potentially its localization. Auditory brainstem response (ABR) analysis demonstrated that mice homozygous for the missense mutation are profoundly deaf, consistent with an essential role for otoferlin in inner hair cell neurotransmission. Vestibular-evoked potentials (VsEPs) of mutant mice, however, were equivalent to those of wildtype mice, indicating that otoferlin is unnecessary for vestibular function even though it is highly expressed in both vestibular and cochlear hair cells.

Targeted knockout and lacZ reporter expression of the mouse Tmhs deafness gene and characterization of the hscy-2J mutation.

The Tmhs gene codes for a tetraspan transmembrane protein that is expressed in hair cell stereocilia. We previously showed that a spontaneous missense mutation of Tmhs underlies deafness and vestibular dysfunction in the hurry-scurry (hscy) mouse. Subsequently, mutations in the human TMHS gene were shown to be responsible for DFNB67, an autosomal recessive nonsyndromic deafness locus. Here we describe a genetically engineered null mutation of the mouse Tmhs gene (Tmhs ( tm1Kjn )) and show that its phenotype is identical to that of the hscy missense mutation, confirming the deleterious nature of the hscy cysteine-to-phenylalanine substitution. In the targeted null allele, the Tmhs promoter drives expression of a lacZ reporter gene. Visualization of beta-galactosidase activity in Tmhs ( tm1Kjn ) heterozygous mice indicates that Tmhs is highly expressed in the cochlear and vestibular hair cells of the inner ear. Expression is first detectable at E15.5, peaks around P0, decreases slightly at P6, and is absent by P15, a duration that supports the involvement of Tmhs in stereocilia development. Tmhs reporter gene expression also was detected in several cranial and cervical sensory ganglia, but not in the vestibular or spiral ganglia. We also describe a new nontargeted mutation of the Tmhs gene, hscy-2J, that causes abnormal splicing from a cryptic splice site within exon 2 and is predicted to produce a functionally null protein lacking 51 amino acids of the wild-type sequence.

The chloride intracellular channel protein CLIC5 is expressed at high levels in hair cell stereocilia and is essential for normal inner ear function.

Although CLIC5 is a member of the chloride intracellular channel protein family, its association with actin-based cytoskeletal structures suggests that it may play an important role in their assembly or maintenance. Mice homozygous for a new spontaneous recessive mutation of the Clic5 gene, named jitterbug (jbg), exhibit impaired hearing and vestibular dysfunction. The jbg mutation is a 97 bp intragenic deletion that causes skipping of exon 5, which creates a translational frame shift and premature stop codon. Western blot and immunohistochemistry results confirmed the predicted absence of CLIC5 protein in tissues of jbg/jbg mutant mice. Histological analysis of mutant inner ears revealed dysmorphic stereocilia and progressive hair cell degeneration. In wild-type mice, CLIC5-specific immunofluorescence was detected in stereocilia of both cochlear and vestibular hair cells and also along the apical surface of Kolliker's organ during cochlear development. Refined immunolocalization in rat and chicken vestibular hair cells showed that CLIC5 is limited to the basal region of the hair bundle, similar to the known location of radixin. Radixin immunostaining appeared reduced in hair bundles of jbg mutant mice. By mass spectrometry and immunoblotting, CLIC5 was shown to be expressed at high levels in stereocilia of the chicken utricle, in an approximate 1:1 molar ratio with radixin. These results suggest that CLIC5 associates with radixin in hair cell stereocilia and may help form or stabilize connections between the plasma membrane and the filamentous actin core.

Editing-defective tRNA synthetase causes protein misfolding and neurodegeneration.

Misfolded proteins are associated with several pathological conditions including neurodegeneration. Although some of these abnormally folded proteins result from mutations in genes encoding disease-associated proteins (for example, repeat-expansion diseases), more general mechanisms that lead to misfolded proteins in neurons remain largely unknown. Here we demonstrate that low levels of mischarged transfer RNAs (tRNAs) can lead to an intracellular accumulation of misfolded proteins in neurons. These accumulations are accompanied by upregulation of cytoplasmic protein chaperones and by induction of the unfolded protein response. We report that the mouse sticky mutation, which causes cerebellar Purkinje cell loss and ataxia, is a missense mutation in the editing domain of the alanyl-tRNA synthetase gene that compromises the proofreading activity of this enzyme during aminoacylation of tRNAs. These findings demonstrate that disruption of translational fidelity in terminally differentiated neurons leads to the accumulation of misfolded proteins and cell death, and provide a novel mechanism underlying neurodegeneration.