Hair Bundle Development Research Articles

Exploring the Heterogeneity of Non-Syndromic Hearing Loss: A Comprehensive Review of Implicated Genes and the Role of Whole Exome Sequencing

Background: Hereditary hearing loss (HHL) accounts for approximately 50-60% of all hearing loss (HL) cases, highlighting the significant role genetics play in this condition. HHL can be mostly classified into two main forms: syndromic and non-syndromic. Of these, non-syndromic hearing loss (NSHL) is the most prevalent, contributing to over 70% of HHL cases. The hearing process involves a complex interplay of various components inside the ear, working together to convert sound waves into comprehensible signals for the brain. Non-syndromic hearing loss (NSHL) is known for its extraordinary diversity and complexity, influenced by a vast array of genes, with more than 120 identified thus far. These genes can be categorized into distinct functional groups, encompassing cochlear ion homeostasis, hair bundle development and cell adhesion, synaptic transmission, transcriptional regulation, and mitochondrial function. The advent of next-generation sequencing (NGS) technologies has been instrumental in unraveling the genetic underpinnings of NSHL. Among NGS techniques, whole exome sequencing (WES) appeared as a revolutionary advancement, outperforming other approaches like targeted panel sequencing and whole-genome sequencing in terms of cost-effectiveness and efficiency. WES not only aids in deciphering the genetic basis of hearing loss but also offers the potential to revolutionize patient care and diagnosis. It paves the way for a new era for precision medicine in the realm of hearing loss, promising more accurate and tailored treatments for affected individuals. This transformative approach is poised to bring about substantial advancements in our understanding of hereditary hearing loss and its clinical management.

The proper timing of Atoh1 expression is pivotal for hair cell subtype differentiation and the establishment of inner ear function

Atoh1 overexpression is essential for hair cell (HC) regeneration in the sensory epithelium of mammalian auditory and vestibular organs. However, Atoh1 overexpression alone cannot induce fully mature and functional HCs in the mammalian inner ear. In the current study, we investigated the effect of Atoh1 constitutive overexpression in native HCs by manipulating Atoh1 expression at different developmental stages. We demonstrated that constitutive overexpression of Atoh1 in native vestibular HCs did not affect cell survival but did impair vestibular function by interfering with the subtype differentiation of HCs and hair bundle development. In contrast, Atoh1 overexpression in cochlear HCs impeded their maturation, eventually leading to gradual HC loss in the cochlea and hearing dysfunction. Our study suggests that time-restricted Atoh1 expression is essential for the differentiation and survival of HCs in the inner ear, and this is pivotal for both hearing and vestibular function re-establishment through Atoh1 overexpression-induced HC regeneration strategies.

Control of stereocilia length during development of hair bundles.

Assembly of the hair bundle, the sensory organelle of the inner ear, depends on differential growth of actin-based stereocilia. Separate rows of stereocilia, labeled 1 through 3 from tallest to shortest, lengthen or shorten during discrete time intervals during development. We used lattice structured illumination microscopy and surface rendering to measure dimensions of stereocilia from mouse apical inner hair cells during early postnatal development; these measurements revealed a sharp transition at postnatal day 8 between stage III (row 1 and 2 widening; row 2 shortening) and stage IV (final row 1 lengthening and widening). Tip proteins that determine row 1 lengthening did not accumulate simultaneously during stages III and IV; while the actin-bundling protein EPS8 peaked at the end of stage III, GNAI3 peaked several days later-in early stage IV-and GPSM2 peaked near the end of stage IV. To establish the contributions of key macromolecular assemblies to bundle structure, we examined mouse mutants that eliminated tip links (Cdh23v2J or Pcdh15av3J), transduction channels (TmieKO), or the row 1 tip complex (Myo15ash2). Cdh23v2J/v2J and Pcdh15av3J/av3J bundles had adjacent stereocilia in the same row that were not matched in length, revealing that a major role of these cadherins is to synchronize lengths of side-by-side stereocilia. Use of the tip-link mutants also allowed us to distinguish the role of transduction from effects of transduction proteins themselves. While levels of GNAI3 and GPSM2, which stimulate stereocilia elongation, were greatly attenuated at the tips of TmieKO/KO row 1 stereocilia, they accumulated normally in Cdh23v2J/v2J and Pcdh15av3J/av3J stereocilia. These results reinforced the suggestion that the transduction proteins themselves facilitate localization of proteins in the row 1 complex. By contrast, EPS8 concentrates at tips of all TmieKO/KO, Cdh23v2J/v2J, and Pcdh15av3J/av3J stereocilia, correlating with the less polarized distribution of stereocilia lengths in these bundles. These latter results indicated that in wild-type hair cells, the transduction complex prevents accumulation of EPS8 at the tips of shorter stereocilia, causing them to shrink (rows 2 and 3) or disappear (row 4 and microvilli). Reduced rhodamine-actin labeling at row 2 stereocilia tips of tip-link and transduction mutants suggests that transduction's role is to destabilize actin filaments there. These results suggest that regulation of stereocilia length occurs through EPS8 and that CDH23 and PCDH15 regulate stereocilia lengthening beyond their role in gating mechanotransduction channels.

Loxhd1 Mutations Cause Mechanotransduction Defects in Cochlear Hair Cells.

Sound detection happens in the inner ear via the mechanical deflection of the hair bundle of cochlear hair cells. The hair bundle is an apical specialization consisting of actin-filled membrane protrusions (called stereocilia) connected by tip links (TLs) that transfer the deflection force to gate the mechanotransduction channels. Here, we identified the hearing loss-associated Loxhd1/DFNB77 gene as being required for the mechanotransduction process. LOXHD1 consists of 15 polycystin lipoxygenase α-toxin (PLAT) repeats, which in other proteins can bind lipids and proteins. LOXHD1 was distributed along the length of the stereocilia. Two LOXHD1 mouse models with mutations in the 10th PLAT repeat exhibited mechanotransduction defects (in both sexes). While mechanotransduction currents in mutant inner hair cells (IHCs) were similar to wild-type levels in the first postnatal week, they were severely affected by postnatal day 11. The onset of the mechanotransduction phenotype was consistent with the temporal progression of postnatal LOXHD1 expression/localization in the hair bundle. The mechanotransduction defect observed in Loxhd1-mutant IHCs was not accompanied by a morphologic defect of the hair bundle or a reduction in TL number. Using immunolocalization, we found that two proteins of the upper and lower TL protein complexes (Harmonin and LHFPL5) were maintained in the mutants, suggesting that the mechanotransduction machinery was present but not activatable. This work identified a novel LOXHD1-dependent step in hair bundle development that is critical for mechanotransduction in mature hair cells as well as for normal hearing function in mice and humans.SIGNIFICANCE STATEMENT Hair cells detect sound-induced forces via the hair bundle, which consists of membrane protrusions connected by tip links. The mechanotransduction machinery forms protein complexes at the tip-link ends. The current study showed that LOXHD1, a multirepeat protein responsible for hearing loss in humans and mice when mutated, was required for hair-cell mechanotransduction, but only after the first postnatal week. Using immunochemistry, we demonstrated that this defect was not caused by the mislocalization of the tip-link complex proteins Harmonin or LHFPL5, suggesting that the mechanotransduction protein complexes were maintained. This work identified a new step in hair bundle development, which is critical for both hair-cell mechanotransduction and hearing.

Grxcr1 Promotes Hair Bundle Development by Destabilizing the Physical Interaction between Harmonin and Sans Usher Syndrome Proteins

SUMMARYMorphogenesis and mechanoelectrical transduction of the hair cell mechanoreceptor depend on the correct assembly of Usher syndrome (USH) proteins into highly organized macromolecular complexes. Defects in these proteins lead to deafness and vestibular areflexia in USH patients. Mutations in a non-USH protein, glutaredoxin domain-containing cysteine-rich 1 (GRXCR1), cause non-syndromic sensorineural deafness. To understand the deglutathionylating enzyme function of GRXCR1 in deafness, we generated two grxcr1 zebrafish mutant alleles. We found that hair bundles are thinner in homozygous grxcr1 mutants, similar to the USH1 mutants ush1c (Harmonin) and ush1ga (Sans). In vitro assays showed that glutathionylation promotes the interaction between Ush1c and Ush1ga and that Grxcr1 regulates mechanoreceptor development by preventing physical interaction between these proteins without affecting the assembly of another USH1 protein complex, the Ush1c-Cadherin23-Myosin7aa tripartite complex. By elucidating the molecular mechanism through which Grxcr1 functions, we also identify a mechanism that dynamically regulates the formation of Usher protein complexes.

Deletion of Kncn Does Not Affect Kinocilium and Stereocilia Bundle Morphogenesis and Mechanotransduction in Cochlear Hair Cells.

Auditory hair cells possess stunning cilia structure that composes of a bundle of stereocilia for mechano-electrical transduction and a single kinocilium for guiding the polarity of hair bundle towards maturation. However, the molecules underlying kinocilium function have not yet been fully understood. Hence, the proteins involved in hair bundle development and function are of a large interest. From a fine microarray analysis, we found that kinocilin (Kncn) was enriched in hair cell specific expression profile. Consistently, it has been reported that KNCN was a protein mainly located in the kinocilium of hair cells in the inner ear. However, the hypothesis that KNCN is a kinocilium protein has not been validated in mice with Kncn gene perturbed. In this study, we generated Kncn knockout mouse lines by CRISPR/Cas9 technique and further examined the morphology and function of cochlear hair cells. Our results showed that there was no obvious hearing loss in the knockout mice, determined by audiometry. Histological study demonstrated that the inner ear and hair cell structure were intact. Especially, there was no deficit of mechanotransduction (MET) in cochlear outer hair cells (OHCs). In summary, our work suggests that KNCN is not essential for kinocilium-oriented hair bundle function in cochlear hair cells.

Annexin A5 is the Most Abundant Membrane-Associated Protein in Stereocilia but is Dispensable for Hair-Bundle Development and Function.

The phospholipid- and Ca2+-binding protein annexin A5 (ANXA5) is the most abundant membrane-associated protein of ~P23 mouse vestibular hair bundles, the inner ear’s sensory organelle. Using quantitative mass spectrometry, we estimated that ANXA5 accounts for ~15,000 copies per stereocilium, or ~2% of the total protein there. Although seven other annexin genes are expressed in mouse utricles, mass spectrometry showed that none were present at levels near ANXA5 in bundles and none were upregulated in stereocilia of Anxa5−/− mice. Annexins have been proposed to mediate Ca2+-dependent repair of membrane lesions, which could be part of the repair mechanism in hair cells after noise damage. Nevertheless, mature Anxa5−/− mice not only have normal hearing and balance function, but following noise exposure, they are identical to wild-type mice in their temporary or permanent changes in hearing sensitivity. We suggest that despite the unusually high levels of ANXA5 in bundles, it does not play a role in the bundle’s key function, mechanotransduction, at least until after two months of age in the cochlea and six months of age in the vestibular system. These results reinforce the lack of correlation between abundance of a protein in a specific compartment or cellular structure and its functional significance.

Developmental regulation of planar cell polarity and hair-bundle morphogenesis in auditory hair cells: lessons from human and mouse genetics.

Hearing loss is the most common and costly sensory defect in humans and genetic causes underlie a significant proportion of affected individuals. In mammals, sound is detected by hair cells (HCs) housed in the cochlea of the inner ear, whose function depends on a highly specialized mechanotransduction organelle, the hair bundle. Understanding the factors that regulate the development and functional maturation of the hair bundle is crucial for understanding the pathophysiology of human deafness. Genetic analysis of deafness genes in animal models, together with complementary forward genetic screens and conditional knock-out mutations in essential genes, have provided great insights into the molecular machinery underpinning hair-bundle development and function. In this review, we highlight recent advances in our understanding of hair-bundle morphogenesis, with an emphasis on the molecular pathways governing hair-bundle polarity and orientation. We next discuss the proteins and structural elements important for hair-cell mechanotransduction as well as hair-bundle cohesion and maintenance. In addition, developmental signals thought to regulate tonotopic features of HCs are introduced. Finally, novel approaches that complement classic genetics for studying the molecular etiology of human deafness are presented. WIREs Dev Biol 2016, 5:85-101. doi: 10.1002/wdev.202 For further resources related to this article, please visit the WIREs website.

The cuticular plate: a riddle, wrapped in a mystery, inside a hair cell.

The mechanosensitive hair cells of the inner ear are crucial to hearing and vestibular function. Each hair cell detects the mechanical stimuli associated with sound or head movement with a hair bundle at the apical surface of the cell, consisting of a precise array of actin-based stereocilia. Each stereocilium inserts as a rootlet into a dense filamentous actin mesh known as the cuticular plate. Disruption of the parallel actin bundles forming the stereocilia results in hearing impairments and balance defects. The cuticular plate is thought to be involved in holding the stereocilia in place. However, the precise role of the cuticular plate in hair bundle development, maintenance, and hearing remains unknown. Ultrastructural studies have revealed a complex cytoskeletal architecture, but a lack of knowledge of proteins that inhabit the cuticular plate and a dearth of mutations that perturb relevant proteins have hindered our understanding of the functions of the cuticular plate. Here, we discuss what is known about the structure and development of this unique and poorly-understood actin-rich organelle.

Identification of a novel compound heterozygous mutation in PTPRQ in a DFNB84 family with prelingual sensorineural hearing impairment.

With a prevalence of 0.1 %, hearing loss is among the most common sensory impairments and affects several million people around the world. Identification of deafness-related genes or loci may facilitate basic research and clinical translational research of the disorder. The PTPRQ gene encodes protein tyrosine phosphatase receptor Q, which is required for the formation of shaft connectors and the normal maturation and development of hair bundles in the mammalian cochlea. Here, we present the genetic and molecular characteristics of a Kazakh family with an autosomal recessive non-syndromic hearing impairment, DFNB84. Using whole-exome sequencing, we identified two mutations that together form a novel compound heterozygous mutation in PTPRQ. Sanger sequencing confirmed that the affected members inherited both the c.16_17insT (L8fsX18) and c.2714delA (E909fsX922) mutations. Both mutations lead to a frameshift and a truncated form of the protein. The novel compound heterozygous mutation co-segregated with hearing loss in this family, and neither of the two mutations was found in 200 healthy Kazakh controls or in any of the public databases. In the study, we identified novel mutations in PTPRQ responsible for DFNB84. This is the third report of PTPRQ mutations involved in deafness and the first report of familial deafness in China. The identification of novel mutations in PTPRQ presented here further confirms the essential role of PTPRQ in hearing development and auditory function. Our data provide additional molecular information for establishing a better genotype-phenotype understanding of DFNB84.

The CD2 isoform of protocadherin-15 is an essential component of the tip-link complex in mature auditory hair cells.

Protocadherin-15 (Pcdh15) is a component of the tip-links, the extracellular filaments that gate hair cell mechano-electrical transduction channels in the inner ear. There are three Pcdh15 splice isoforms (CD1, CD2 and CD3), which only differ by their cytoplasmic domains; they are thought to function redundantly in mechano-electrical transduction during hair-bundle development, but whether any of these isoforms composes the tip-link in mature hair cells remains unknown. By immunolabelling and both morphological and electrophysiological analyses of post-natal hair cell-specific conditional knockout mice (Pcdh15ex38-fl/ex38-fl Myo15-cre+/−) that lose only this isoform after normal hair-bundle development, we show that Pcdh15-CD2 is an essential component of tip-links in mature auditory hair cells. The finding, in the homozygous or compound heterozygous state, of a PCDH15 frameshift mutation (p.P1515Tfs*4) that affects only Pcdh15-CD2, in profoundly deaf children from two unrelated families, extends this conclusion to humans. These results provide key information for identification of new components of the mature auditory mechano-electrical transduction machinery. This will also serve as a basis for the development of gene therapy for deafness caused by PCDH15 defects.

An ENU-Induced Mutation of Cdh23 Causes Congenital Hearing Loss, but No Vestibular Dysfunction, in Mice

Mutations in the human cadherin 23 (CDH23) gene cause deafness, neurosensory, autosomal recessive 12 (DFNB12) nonsyndromic hearing loss or Usher syndrome, type 1D (characterized by hearing impairment, vestibular dysfunction, and visual impairment). Reported waltzer mouse strains each harbor a Cdh23-null mutation and present with hearing loss and vestibular dysfunction. Two additional Cdh23 mouse mutants, salsa and erlong, each carry a homozygous Cdh23 missense mutation and have progressive hearing loss. We report the identification of a novel mouse strain, jera, with inherited hearing loss caused by an N-ethyl-N-nitrosourea-induced c.7079T>A mutation in the Cdh23 gene. The mutation generates a missense change, p.V2360E, in Cdh23. Affected mice have profound sensorineural deafness, with no vestibular dysfunction. The p.V2360E mutation is semidominant because heterozygous mice have milder and more progressive hearing loss in advanced age. The mutation affects a highly conserved Ca(2+)-binding motif in extracellular domain 22, thought to be important for Cdh23 structure and dimerization. Molecular modeling suggests that the Cdh23(V2360E/V2360E) mutation alters the structural conformation of the protein and affects Ca(2+)-binding properties. Similar to salsa mice, but in contrast to waltzer mice, hair bundle development is normal in jera and hearing loss appears to be due to the loss of tip links. Thus, jera is a novel mouse model for DFNB12.

Eps8 regulates hair bundle length and functional maturation of mammalian auditory hair cells.

Hair cells of the mammalian cochlea are specialized for the dynamic coding of sound stimuli. The transduction of sound waves into electrical signals depends upon mechanosensitive hair bundles that project from the cell's apical surface. Each stereocilium within a hair bundle is composed of uniformly polarized and tightly packed actin filaments. Several stereociliary proteins have been shown to be associated with hair bundle development and function and are known to cause deafness in mice and humans when mutated. The growth of the stereociliar actin core is dynamically regulated at the actin filament barbed ends in the stereociliary tip. We show that Eps8, a protein with actin binding, bundling, and barbed-end capping activities in other systems, is a novel component of the hair bundle. Eps8 is localized predominantly at the tip of the stereocilia and is essential for their normal elongation and function. Moreover, we have found that Eps8 knockout mice are profoundly deaf and that IHCs, but not OHCs, fail to mature into fully functional sensory receptors. We propose that Eps8 directly regulates stereocilia growth in hair cells and also plays a crucial role in the physiological maturation of mammalian cochlear IHCs. Together, our results indicate that Eps8 is critical in coordinating the development and functionality of mammalian auditory hair cells.

How the Genetics of Deafness Illuminates Auditory Physiology

Although the basic principles underlying the function of the peripheral auditory system have been known for many years, the molecules required for hearing have hitherto remained elusive. Genetic approaches have recently provided unparalleled molecular insight into how the hair bundle, the hair cell's mechanosensory organelle, forms and functions. We discuss how the proteins encoded by the Usher syndrome type 1 genes form molecular complexes required for hair-bundle development and for gating the mechanotransducer channel. We show how mouse models for nonsyndromic forms of deafness involving genes encoding Triobp and stereocilin reveal, respectively, the way stereocilia rootlets contribute to the hair bundle's mechanical properties and how the hair bundle produces suppressive masking, a property that contributes to speech intelligibility. Finally, we examine how mutations in the genes encoding α- and β-tectorin reveal multiple roles for the tectorial membrane, an extracellular matrix unique to the cochlea, in stimulating hair bundles.

Linking genes underlying deafness to hair-bundle development and function

The identification of genes underlying monogenic, early-onset forms of deafness in humans has provided unprecedented insight into the molecular mechanisms of hearing in the peripheral auditory system. The molecules involved in the development and function of the cochlea eluded characterization until recently due to the paucity of the principle cell types present in cochlear hair cells, yet a genetic approach has circumvented this problem and succeeded in identifying proteins and deciphering some of the molecular complexes that operate in these cells . In combination with mouse models, the genetic approach is now revealing some of the principles underlying the development and physiology of the cochlea. The review centers on this facet of the genetics of hearing. Focusing on the hair bundle, the mechanosensory device of the sensory hair cell, we highlight recent advances in understanding the way in which the hair bundle is formed, how it operates as a mechanotransducer and how it processes sound. In particular, we discuss how this work highlights the roles played by various hair-bundle link types.

Physical and Functional Interaction between Protocadherin 15 and Myosin VIIa in Mechanosensory Hair Cells

Hair cells of the mammalian inner ear are the mechanoreceptors that convert sound-induced vibrations into electrical signals. The molecular mechanisms that regulate the development and function of the mechanically sensitive organelle of hair cells, the hair bundle, are poorly defined. We link here two gene products that have been associated with deafness and hair bundle defects, protocadherin 15 (PCDH15) and myosin VIIa (MYO7A), into a common pathway. We show that PCDH15 binds to MYO7A and that both proteins are expressed in an overlapping pattern in hair bundles. PCDH15 localization is perturbed in MYO7A-deficient mice, whereas MYO7A localization is perturbed in PCDH15-deficient mice. Like MYO7A, PCDH15 is critical for the development of hair bundles in cochlear and vestibular hair cells, controlling hair bundle morphogenesis and polarity. Cochlear and vestibular hair cells from PCDH15-deficient mice also show defects in mechanotransduction. Together, our findings suggest that PCDH15 and MYO7A cooperate to regulate the development and function of the mechanically sensitive hair bundle.

Sustained cadherin 23 expression in young and adult cochlea of normal and hearing-impaired mice

Cadherin 23 encodes a single-pass transmembrane protein with 27 extracellular cadherin-domains and localizes to stereocilia where it functions as an inter-stereocilia link. Cadherin 23-deficient mice show congenital deafness in combination with circling behavior as a result of organizational defects in the stereocilia hair bundle; common inbred mouse strains carrying the hypomorphic Cdh23 753 A allele are highly susceptible to sensorineural hearing loss. Here, we show that an antibody (N1086) directed against the intracellular carboxyterminus reacts specifically with cadherin 23 and detects with high sensitivity the isoform devoid of the peptide encoded by exon 68 (CDH23Δ68). Cochlea, vestibule, eye, brain and testis produce the CDH23Δ68 isoform in abundance and form moieties with different molecular weight due to variations in glycosylation content. In the cochlea, CDH23Δ68 expression is highest at postnatal day 1 (P1) and P7; expression is down regulated through P14 and P21 and persists at a low steady-state level throughout adulthood (P160). Furthermore, CDH23Δ68 expression levels in young and adult cochlea are similar among normal and hearing deficient strains (C3HeB/FeJ, C57BL/6J and BUB/BnJ). Finally, by immunofluorescence using an antibody (Pb240) specific for ectodomain 14, we show that cadherin 23 localizes to stereocilia during hair bundle development in late gestation and early postnatal days. Cadherin 23-specific labeling becomes weaker as the hair bundle matures but faint labeling concentrated near the top of stereocilia is still detectable at P35. No labeling of cochlea stereocilia was observed with N1086. In conclusion, our data describe a cadherin 23-specific antibody with high affinity to the CDH23Δ68 isoform, reveal a dynamic cochlea expression and localization profile and show sustained cadherin 23 levels in adult cochlea of normal and hearing-impaired mice.

Spatiotemporal pattern and isoforms of cadherin 23 in wild type and waltzer mice during inner ear hair cell development

Mutant alleles of the gene encoding cadherin 23 are associated with Usher syndrome type 1 (USH1D), isolated deafness (DFNB12) in humans, and deafness and circling behavior in waltzer ( v) mice. Stereocilia of waltzer mice are disorganized and the kinocilia misplaced, indicating the importance of cadherin 23 for hair bundle development. Cadherin 23 was localized to developing stereocilia and proposed as a component of the tip link. We show that, during development of the inner ear, cadherin 23 is initially detected in centrosomes at E14.5, then along the length of emerging stereocilia, and later becomes concentrated at and subsequently disappears from the tops of stereocilia. In mature vestibular hair bundles, cadherin 23 is present along the kinocilium and in the region of stereocilia–kinocilium bonds, a pattern conserved in mammals, chicks, and frogs. Cadherin 23 is also present in Reissner's membrane (RM) throughout development. In homozygous v 6J mice, a reported null allele, cadherin 23 was absent from stereocilia, but present in kinocilia, RM, and centrosomes. We reconciled these results by identifying two novel isoforms of Cdh23 unaffected in sequence and expression by the v 6J allele. Our results suggest that Cdh23 participation in stereocilia links may be restricted to developing hair bundles.