Anti-apoptotic factor z-Val-Ala-Asp-fluoromethylketone promotes the survival of cochlear hair cells in a mouse model for human deafness.

A major challenge in the inner ear research field is to restore hearing loss of both non-genetic and genetic origin. A large effort is being made to protect hair cells from cell death after exposure to noise or drugs that can cause hearing loss. Our research focused on protecting hair cells from cell death occurring in a genetic model for human deafness. POU4F3 is a transcription factor associated with human hearing impairment. Pou4f3 knockout mice (Pou4f3(-/-)) have no cochlear hair cells, resulting in complete deafness. Although the hair cells appear to form properly, they progressively degenerate via apoptosis. In order to rescue the hair cells in the knockout mice, we produced explant cultures from mouse cochleae at an early embryonic stage and treated the cells with z-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), a general caspase inhibitor. Hair cell numbers in the knockout mice treated with z-VAD-fmk were significantly higher than in the untreated mice. We found that the time window that z-VAD-fmk has a protective effect is between E14.5 (P=0.001) to E16.5 (P=0.03), but not after E18.5. The source of the surviving hair cells is not due to proliferation, as measured by 5-bromo-2-deoxyuridine (BrdU) labeling, or to supporting cell transdifferentiation to hair cells, since there was no change in supporting cell numbers. Instead, the survival appears to be a direct effect of the anti-apoptotic agent on the dying hair cells with an early developmental window. These results help towards providing a comprehensive understanding of the molecular mechanisms of hair cell death, which might lead to the development of new therapeutic anti-apoptotic agents to alleviate hereditary hearing loss (HL).

MYO6, the human homologue of the gene responsible for deafness in Snell's waltzer mice, is mutated in autosomal dominant nonsyndromic hearing loss.

Mutations in the unconventional myosin VI gene, Myo6, are associated with deafness and vestibular dysfunction in the Snell's waltzer (sv) mouse. The corresponding human gene, MYO6, is located on chromosome 6q13. We describe the mapping of a new deafness locus, DFNA22, on chromosome 6q13 in a family affected by a nonsyndromic dominant form of deafness (NSAD), and the subsequent identification of a missense mutation in the MYO6 gene in all members of the family with hearing loss.

Connexin 31 (GJB3) is expressed in the peripheral and auditory nerves and causes neuropathy and hearing impairment.

Mutations in the connexin 31 (GJB3) gene have been found in subjects with dominant and recessive deafness and in patients with erythrokeratodermia variabilis. We report here a dominant mutation in the GJB3 gene (D66del) in a family affected with peripheral neuropathy and sensorineural hearing impairment. A wide range of disease severity for peripheral neuropathy, from asymptomatic cases to subjects with chronic skin ulcers in their feet and osteomyelitis leading to amputations, was detected in D66del patients. Mild, often asymmetrical, hearing impairment was found in all but one patient with mutation D66del of this family and the same mutation was present in an independent family ascertained because of hearing impairment. We have found mouse connexin 31 (Gjb3) gene expression in the cochlea and in the auditory and sciatic nerves, showing a pattern similar to that of Gjb1 (connexin 32), of which the human ortholog (GJB1) is involved in X-linked peripheral neuropathy. This expression pattern, together with auditory-evoked brainstem anomalous response in D66del patients, indicates that hearing impairment due to GJB3 mutations involves alterations in both the cochlea and the auditory nerve. Peripheral neuropathy is the third phenotypic alteration linked to GJB3 mutations, which enlarges the list of genes that cause this group of heterogeneous disorders.

The Notch ligand Jagged1 is required for inner ear sensory development.

Within the mammalian inner ear there are six separate sensory regions that subserve the functions of hearing and balance, although how these sensory regions become specified remains unknown. Each sensory region is populated by two cell types, the mechanosensory hair cell and the supporting cell, which are arranged in a mosaic in which each hair cell is surrounded by supporting cells. The proposed mechanism for creating the sensory mosaic is lateral inhibition mediated by the Notch signaling pathway. However, one of the Notch ligands, Jagged1 (Jag1), does not show an expression pattern wholly consistent with a role in lateral inhibition, as it marks the sensory patches from very early in their development--presumably long before cells make their final fate decisions. It has been proposed that Jag1 has a role in specifying sensory versus nonsensory epithelium within the ear [Adam, J., Myat, A., Roux, I. L., Eddison, M., Henrique, D., Ish-Horowicz, D. & Lewis, J. (1998) Development (Cambridge, U.K.) 125, 4645--4654]. Here we provide experimental evidence that Notch signaling may be involved in specifying sensory regions by showing that a dominant mouse mutant headturner (Htu) contains a missense mutation in the Jag1 gene and displays missing posterior and sometimes anterior ampullae, structures that house the sensory cristae. Htu/+ mutants also demonstrate a significant reduction in the numbers of outer hair cells in the organ of Corti. Because lateral inhibition mediated by Notch predicts that disruptions in this pathway would lead to an increase in hair cells, we believe these data indicate an earlier role for Notch within the inner ear.

Modifying with mitochondria.

An elegant set of mouse crosses has been used to identify a mitochondrial variant that interacts with a nuclear locus on chromosome 10, Ahl, to modify age-related hearing loss. This discovery sets the stage for the identification of factors that modify expression levels and variability of human hearing impairments.

Inherited connexin mutations associated with hearing loss.

One of the most dramatic discoveries in the field of hereditary hearing loss is the association of this sensory defect with connexin mutations. Most significant is the large proportion, 30-50%, of inherited hearing loss that is due to mutations in connexin 26. The proteins these genes encode are expressed in the cochlear duct, in regions containing gap junctions. Together, these findings suggest a crucial role for gap junction proteins in the mammalian inner ear. Mouse models with specific connexin mutations leading to deafness will help resolve the many questions regarding the role of these gap junction proteins in the inner ear.

Auditory and vestibular mouse mutants: models for human deafness.

We have shown here several examples of how hearing and vestibular impaired mouse mutants are generated and the insight that they provide in the study of auditory and vestibular function. These types of genetic studies may also lead to the identification of disease-susceptibility genes, perhaps the most critical element in presbyacusis (age-related hearing loss). Some individuals may be more prone to hearing loss with increasing age or upon exposure to severe noise, and susceptibility genes may be involved. Different inbred mice show a variety of age-related and noise-induced hearing loss that varies between normal hearing and severe deafness throughout their life span /27/. Genetic diversity between inbred mouse strains has been shown to be a powerful tool for the discovery of modifier genes. Already two studies have found regions in which modifier genes for deafness may reside /28-29/. Future studies will hopefully lead to the identification of genes that modify hearing loss and will help us understand the variability that exists in human hearing, a crucial component in developing successful treatment strategies. The first human non-syndromic deafness-causing gene was identified in 1995, and since then, additional genes have been discovered. Much of the credit for this boom is due to deaf and vestibular mouse mutants. Their study has led to great insight regarding the development and function of the mammalian inner ear, and correlations with human deafness can now be made since mutations in the same genes have been found in these two mammals. As deafness is the most common form of sensory impairment and affects individuals of all ages, elucidating the function of the auditory and vestibular systems through genetic approaches is essential in improving and designing effective treatments for hearing loss.

The prevalence and expression of inherited connexin 26 mutations associated with nonsyndromic hearing loss in the Israeli population.

Connexin 26 (GJB2) mutations lead to hearing loss in a significant proportion of all populations studied so far, despite the fact that at least 50 other genes are also associated with hearing loss. The entire coding region of connexin 26 was sequenced in 75 hearing impaired children and adults in Israel in order to determine the percentage of hearing loss attributed to connexin 26 and the types of mutations in this population. Age of onset in the screened population was both prelingual and postlingual, with hearing loss ranging from moderate to profound. Almost 39% of all persons tested harbored GJB2 mutations, the majority of which were 35delG and 167delT mutations. A novel mutation, involving both a deletion and insertion, 51del12insA, was identified in a family originating from Uzbekistan. Several parameters were examined to establish whether genotype-phenotype correlations exist, including age of onset, severity of hearing loss and audiological characteristics, including pure-tone audiometry, tympanometry, auditory brainstem response (ABR), and transient evoked otoacoustic emissions (TEOAE). All GJB2 mutations were associated with prelingual hearing loss, though severity ranged from moderate to profound, with variability even among hearing impaired siblings. We have not found a significant difference in hearing levels between individuals with 35delG and 167delT mutations. Our results suggest that, in Israel, clinicians should first screen for the common 167delT and 35delG mutations by simple and inexpensive restriction enzyme analysis, although if these are not found, sequencing should be done to rule out additional mutations due to the ethnic diversity in this region.

Clinical characterization of genetic hearing loss caused by a mutation in the POU4F3 transcription factor.

OBJECTIVES: To describe the detailed auditory phenotype of DFNA15, genetic hearing loss associated with a mutation in the POU4F3 transcription factor, and to define genotype-phenotype correlations, namely, how specific mutations lead to particular clinical consequences. DESIGN: An analysis of clinical features of hearing-impaired members of an Israeli family, family H, with autosomal dominant-inherited hearing loss. SETTING: Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel; Department of Audiology, Rabin Medical Center, Petah Tiqwa, Israel; and audiological centers. PARTICIPANTS: Clinical features of 11 affected and 5 unaffected individuals older than 40 years from family H were studied. Mutation analysis was performed in 6 presymptomatic individuals younger than 30 years; clinical features were analyzed in 4 of these family H members. INTERVENTIONS: Hearing was measured by pure-tone audiometry and speech audiometry on all participating relatives of family H. Immittance testing (tympanometry and acoustic reflexes), auditory brainstem response, and otoacoustic emissions were done in a selected patient population. RESULTS: The patients presented with progressive high-tone sensorineural hearing impairment, which became apparent between ages 18 and 30 years. The hearing impairment became more severe with time, eventually causing significant hearing loss across the spectrum at all frequencies. CONCLUSIONS: Our results indicate that POU4F3 mutation-associated deafness cannot be identified through clinical evaluation, but only through molecular analysis. Intrafamilial variability suggests that other genetic or environmental factors may modify the age at onset and rate of progression.

Genomic structure of the human unconventional myosin VI gene.

Mutations in myosin VI (Myo6) cause deafness and vestibular dysfunction in Snell's waltzer mice. Mutations in two other unconventional myosins cause deafness in both humans and mice, making myosin VI an attractive candidate for human deafness. In this report, we refined the map position of human myosin VI (MYO6) by radiation hybrid mapping and characterized the genomic structure of myosin VI. Human myosin VI is composed of 32 coding exons, spanning a genomic region of approximately 70 kb. Exon 30, containing a putative CKII site, was found to be alternatively spliced and appears only in fetal and adult human brain. D6S280 and D6S284 flank the myosin VI gene and were used to screen hearing impaired sib pairs for concordance with the polymorphic markers. No disease-associated mutations were identified in twenty-five families screened for myosin VI mutations by SSCP analysis. Three coding single nucleotide polymorphisms (cSNPs) were identified in myosin VI that did not alter the amino acid sequence. Myosin VI mutations may be rare in the human deaf population or alternatively, may be found in a population not yet examined. The determination of the MYO6 genomic structure will enable screening of individuals with non-syndromic deafness, Usher's syndrome, or retinopathies associated with human chromosome 6q for mutations in this unconventional myosin.

Role of myosin VI in the differentiation of cochlear hair cells.

The mouse mutant Snell's waltzer (sv) has an intragenic deletion of the Myo6 gene, which encodes the unconventional myosin molecule myosin VI (K. B. Avraham et al., 1995, Nat. Genet. 11, 369-375). Snell's waltzer mutants exhibit behavioural abnormalities suggestive of an inner ear defect, including lack of responsiveness to sound, hyperactivity, head tossing, and circling. We have investigated the effects of a lack of myosin VI on the development of the sensory hair cells of the cochlea in these mutants. In normal mice, the hair cells sprout microvilli on their upper surface, and some of these grow to form a crescent or V-shaped array of modified microvilli, the stereocilia. In the mutants, early stages of stereocilia development appear to proceed normally because at birth many stereocilia bundles have a normal appearance, but in places there are signs of disorganisation of the bundles. Over the next few days, the stereocilia become progressively more disorganised and fuse together. Practically all hair cells show fused stereocilia by 3 days after birth, and there is extensive stereocilia fusion by 7 days. By 20 days, giant stereocilia are observed on top of the hair cells. At 1 and 3 days after birth, hair cells of mutants and controls take up the membrane dye FM1-43, suggesting that endocytosis occurs in mutant hair cells. One possible model for the fusion is that myosin VI may be involved in anchoring the apical hair cell membrane to the underlying actin-rich cuticular plate, and in the absence of normal myosin VI this apical membrane will tend to pull up between stereocilia, leading to fusion.

Tailchaser (Tlc): a new mouse mutation affecting hair bundle differentiation and hair cell survival.

We have undertaken a phenotypic approach in the mouse to identifying molecules involved in inner ear function by N-ethyl-N-nitrosourea mutagenesis followed by screening for new dominant mutations affecting hearing or balance. The pathology and genetic mapping of the first of these new mutants, tailchaser (Tlc), is described here. Tlc/+ mutants display classic behavioural symptoms of a vestibular dysfunction, including head-shaking and circling. Behavioural testing of ageing mice revealed a gradual deterioration of both hearing and balance function, indicating that the pathology caused by the Tlc mutation is progressive, similar to many dominant nonsyndromic deafnesses in humans. Based on scanning electron microscopy (SEM) studies, Tlc clearly plays a developmental role in the hair cells of the cochlea since the stereocilia bundles fail to form the characteristic V-shape pattern around the time of birth. By young adult stages, Tlc/+ outer hair bundles are grossly disorganised although inner hair bundles appear relatively normal by SEM. Increased compound action potential thresholds revealed that the Tlc/+ cochlear hair cells were not functioning normally in young adults. Similar to inner hair cells, the hair bundles of the vestibular hair cells also do not appear grossly disordered. However, all types of hair cells in the Tlc/+ inner ear eventually degenerate, apparently regardless of the degree of organisation of their hair bundles. We have mapped the Tlc mutation to a 12 cM region of chromosome 2, between D2Mit164 and D2Mit423. Based on the mode of inheritance and map location, Tlc appears to be a novel mouse mutation affecting both hair cell survival and stereocilia bundle development.