Gwenaëlle S. G. Géléoc

TRPA1 is a candidate for the mechanosensitive transduction channel of vertebrate hair cells.

Mechanical deflection of the sensory hair bundles of receptor cells in the inner ear causes ion channels located at the tips of the bundle to open, thereby initiating the perception of sound. Although some protein constituents of the transduction apparatus are known, the mechanically gated transduction channels have not been identified in higher vertebrates. Here, we investigate TRP (transient receptor potential) ion channels as candidates and find one, TRPA1 (also known as ANKTM1), that meets criteria for the transduction channel. The appearance of TRPA1 messenger RNA expression in hair cell epithelia coincides developmentally with the onset of mechanosensitivity. Antibodies to TRPA1 label hair bundles, especially at their tips, and tip labelling disappears when the transduction apparatus is chemically disrupted. Inhibition of TRPA1 protein expression in zebrafish and mouse inner ears inhibits receptor cell function, as assessed with electrical recording and with accumulation of a channel-permeant fluorescent dye. TRPA1 is probably a component of the transduction channel itself.

Differential Distribution of Stem Cells in the Auditory and Vestibular Organs of the Inner Ear

The adult mammalian cochlea lacks regenerative capacity, which is the main reason for the permanence of hearing loss. Vestibular organs, in contrast, replace a small number of lost hair cells. The reason for this difference is unknown. In this work we show isolation of sphere-forming stem cells from the early postnatal organ of Corti, vestibular sensory epithelia, the spiral ganglion, and the stria vascularis. Organ of Corti and vestibular sensory epithelial stem cells give rise to cells that express multiple hair cell markers and express functional ion channels reminiscent of nascent hair cells. Spiral ganglion stem cells display features of neural stem cells and can give rise to neurons and glial cell types. We found that the ability for sphere formation in the mouse cochlea decreases about 100-fold during the second and third postnatal weeks; this decrease is substantially faster than the reduction of stem cells in vestibular organs, which maintain their stem cell population also at older ages. Coincidentally, the relative expression of developmental and progenitor cell markers in the cochlea decreases during the first 3 postnatal weeks, which is in sharp contrast to the vestibular system, where expression of progenitor cell markers remains constant or even increases during this period. Our findings indicate that the lack of regenerative capacity in the adult mammalian cochlea is either a result of an early postnatal loss of stem cells or diminishment of stem cell features of maturing cochlear cells.

Mechanotransduction in mouse inner ear hair cells requires transmembrane channel–like genes

Inner ear hair cells convert the mechanical stimuli of sound, gravity, and head movement into electrical signals. This mechanotransduction process is initiated by opening of cation channels near the tips of hair cell stereocilia. Since the identity of these ion channels is unknown, and mutations in the gene encoding transmembrane channel-like 1 (TMC1) cause hearing loss without vestibular dysfunction in both mice and humans, we investigated the contribution of Tmc1 and the closely related Tmc2 to mechanotransduction in mice. We found that Tmc1 and Tmc2 were expressed in mouse vestibular and cochlear hair cells and that GFP-tagged TMC proteins localized near stereocilia tips. Tmc2 expression was transient in early postnatal mouse cochlear hair cells but persisted in vestibular hair cells. While mice with a targeted deletion of Tmc1 (Tmc1(Δ) mice) were deaf and those with a deletion of Tmc2 (Tmc2(Δ) mice) were phenotypically normal, Tmc1(Δ)Tmc2(Δ) mice had profound vestibular dysfunction, deafness, and structurally normal hair cells that lacked all mechanotransduction activity. Expression of either exogenous TMC1 or TMC2 rescued mechanotransduction in Tmc1(Δ)Tmc2(Δ) mutant hair cells. Our results indicate that TMC1 and TMC2 are necessary for hair cell mechanotransduction and may be integral components of the mechanotransduction complex. Our data also suggest that persistent TMC2 expression in vestibular hair cells may preserve vestibular function in humans with hearing loss caused by TMC1 mutations.

TMC1 and TMC2 Are Components of the Mechanotransduction Channel in Hair Cells of the Mammalian Inner Ear

Sensory transduction in auditory and vestibular hair cells requires expression of transmembrane channel-like (Tmc) 1 and 2 genes, but the function of these genes is unknown. To investigate the hypothesis that TMC1 and TMC2 proteins are components of the mechanosensitive ion channels that convert mechanical information into electrical signals, we recorded whole-cell and single-channel currents from mouse hair cells that expressed Tmc1, Tmc2, or mutant Tmc1. Cells that expressed Tmc2 had high calcium permeability and large single-channel currents, while cells with mutant Tmc1 had reduced calcium permeability and reduced single-channel currents. Cells that expressed Tmc1 and Tmc2 had a broad range of single-channel currents, suggesting multiple heteromeric assemblies of TMC subunits. The data demonstrate TMC1 and TMC2 are components of hair cell transduction channels and contribute to permeation properties. Gradients in TMC channel composition may also contribute to variation in sensory transduction along the tonotopic axis of the mammalian cochlea.

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.

Developmental acquisition of sensory transduction in hair cells of the mouse inner ear

Sensory transduction in hair cells requires assembly of membrane-bound transduction channels, extracellular tip-links and intracellular adaptation motors with sufficient precision to confer nanometer displacement sensitivity. Here we present evidence based on FM1-43 fluorescence, scanning electron microscopy and RT-PCR that these three essential elements are acquired concurrently between embryonic day 16 and 17, several days after the appearance of hair bundles, and that their acquisition coincides with the onset of mechanotransduction.

Tonotopic Gradient in the Developmental Acquisition of Sensory Transduction in Outer Hair Cells of the Mouse Cochlea

Inner ear hair cells are exquisite mechanosensors that transduce nanometer scale deflections of their sensory hair bundles into electrical signals. Several essential elements must be precisely assembled during development to confer the unique structure and function of the mechanotransduction apparatus. Here we investigated the functional development of the transduction complex in outer hair cells along the length of mouse cochlea acutely excised between embryonic day 17 (E17) and postnatal day 8 (P8). We charted development of the stereociliary bundle using scanning electron microscopy; FM1-43 uptake, which permeates hair cell transduction channels, mechanotransduction currents evoked by rapid hair bundle deflections, and mRNA expression of possible components of the transduction complex. We demonstrated that uptake of FM1-43 first occurred in the basal portion of the cochlea at P0 and progressed toward the apex over the subsequent week. Electrophysiological recordings obtained from 234 outer hair cells between E17 and P8 from four cochlear regions revealed a correlation between the pattern of FM1-43 uptake and the acquisition of mechanotransduction. We found a spatiotemporal gradient in the properties of transduction including onset, amplitude, operating range, time course, and extent of adaptation. We used quantitative RT-PCR to examine relative mRNA expression of several hair cell myosins and candidate tip-link molecules. We found spatiotemporal expression patterns for mRNA that encodes cadherin 23, protocadherin 15, myosins 3a, 7a, 15a, and PMCA2 that preceded the acquisition of transduction. The spatiotemporal expression patterns of myosin 1c and PMCA2 mRNA were correlated with developmental changes in several properties of mechanotransduction.

Sound Strategies for Hearing Restoration

Background Sensorineural hearing loss is the most common sensory deficit in the world, with nearly 300 million affected individuals. The problem is multifactorial and can arise from damage or death of the primary sensory cells or the inner-ear neurons that relay auditory information to the brain. The inner-ear sensory cells and neurons can be damaged by environmental insult (such as exposure to infectious agents, drugs such as aminoglycoside antibiotics or chemotherapeutics, or overexposure to loud sounds) or by a host of genetic factors. Because mature inner ears lack the capacity for self-repair, the cellular damage is permanent. In addition to acquired hearing loss, more than 300 genetic loci have been linked to hereditary hearing loss, with about 70 of the causative genes identified. Eighty percent of genetic hearing loss is recessive, with the rest inherited as a dominant trait. More subtle genetic defects that cause a predisposition toward age-related hearing loss are poorly understood. Unfortunately, there is no cure for acquired, inherited, or age-related hearing loss. Native and engineered mechanosensory hair cells of the mammalian inner ear. Airborne sound vibrations, funneled through the external ear, are relayed through the middle ear to the inner ear, where they propagate as sound pressure waves in the fluid-filled spaces of the cochlea. The human cochlea contains ~16,000 sensory hair cells that convert sound pressure into electrical signals. (A) Hair cells are crowned with mechanosensory organelles composed of 50 to 100 actin-filled microvilli, organized into staircase arrays known as hair bundles. Extracellular linkages couple microvilli and ensure that the entire bundle responds to sound stimulation with coherent motion. (B) Bundle motion focuses sound energy onto the mechanotransduction apparatus, which includes one or two mechanically gated ion channels (50 to 100 per cell). According to the prevailing model, the channel complex is anchored to the actin core on the intracellular side at the tip of the shorter microvillus. On the extracellular side, there is a tip-link that extends to the side of the adjacent taller neighbor. At the top end, the tip-link is tethered to intracellular motor molecules that regulate tension within the apparatus. Sound-induced changes in tip-link tension modulate the open probability of the mechanosensitive channels, which in turn modulate the flow of electric current into the cell. Disruption of the sensory hair bundle by genetic mutation, loud sounds, or ototoxic drugs can lead to hair-cell dysfunction and deafness. Strategies to restore hair cells and mechanosensory function are being developed to treat patients with neurosensory hearing loss. [Illustrated by B. Pan] (C) Scanning electron microscopy image of a native hair bundle from the mouse inner ear. [Reprinted from J. R. Holt et al., Cell 108, 371–381 (2002) by permission from Elsevier] (D) A hair-cell–like cell generated via a gene-therapy strategy and viral-mediated delivery of the transcription factor Atoh1 into the inner ear of a deaf guinea pig. [Reprinted from Kawamoto et al., J. Neurosci. 23, 4395–4400 (2003) by permission of the Society for Neuroscience] (E) A hair-cell–like cell generated from embryonic stem cells and a stepwise differentiation protocol. Similar cells generate functional mechanosensitive responses to microvilli deflection. [Reprinted from Oshima et al., Cell 141, 704–716 (2010) by permission from Elsevier] Advances Efforts to restore and repair damaged inner-ear cells have intensified over the past 10 years. Thus far, a major thrust has been to adapt three biological strategies for use in the inner ear: gene therapy, stem-cell therapy, and molecular therapy. Using these approaches, researchers have restored sensory function at the cellular level in animal models of human hearing loss. A few reports suggest functional recovery at the systems and behavioral levels, although caveats remain. Outlook As the population continues to age and expand, so will the number of patients who suffer from clinically serious hearing loss. As such, the need for a deeper and comprehensive understanding of hearing-loss therapies is more pressing than ever. Although the pace of progress is accelerating and clinical trials are on the horizon, it is clear that there are still a number of hurdles to overcome. Overall, there are reasons to be both cautious and optimistic as we attempt to repair and regenerate one of nature’s most exquisite mechanosensory devices: the human inner ear. Hearing Aid Many millions of people across the globe are subject to hearing loss. Géléoc and Holt (10.1126/science.1241062) review recent developments in potential therapeutic strategies to restore inner-ear function in patients with acquired or genetic forms of deafness. While challenges remain, fundamental research in molecular, gene, and stem-cell therapies has enabled progress toward developing alternatives to conventional, sound amplification–based prostheses. Hearing loss is the most common sensory deficit in humans, with some estimates suggesting up to 300 million affected individuals worldwide. Both environmental and genetic factors contribute to hearing loss and can cause death of sensory cells and neurons. Because these cells do not regenerate, the damage tends to accumulate, leading to profound deafness. Several biological strategies to restore auditory function are currently under investigation. Owing to the success of cochlear implants, which offer partial recovery of auditory function for some profoundly deaf patients, potential biological therapies must extend hearing restoration to include greater auditory acuity and larger patient populations. Here, we review the latest gene, stem-cell, and molecular strategies for restoring auditory function in animal models and the prospects for translating these approaches into viable clinical therapies.

Developmental Acquisition of Voltage-Dependent Conductances and Sensory Signaling in Hair Cells of the Embryonic Mouse Inner Ear

How and when sensory hair cells acquire the remarkable ability to detect and transmit mechanical information carried by sound and head movements has not been illuminated. Previously, we defined the onset of mechanotransduction in embryonic hair cells of mouse vestibular organs to be at approximately embryonic day 16 (E16). Here we examine the functional maturation of hair cells in intact sensory epithelia excised from the inner ears of embryonic mice. Hair cells were studied at stages between E14 and postnatal day 2 using the whole-cell, tight-seal recording technique. We tracked the developmental acquisition of four voltage-dependent conductances. We found a delayed rectifier potassium conductance that appeared as early as E14 and grew in amplitude over the subsequent prenatal week. Interestingly, we also found a low-voltage-activated potassium conductance present at E18, ∼1 week earlier than reported previously. An inward rectifier conductance appeared at approximately E15 and doubled in size over the next few days. We also noted transient expression of a voltage-gated sodium conductance that peaked between E16 and E18 and then declined to near zero at birth. We propose that hair cells undergo a stereotyped developmental pattern of ion channel acquisition and that the precise pattern may underlie other developmental processes such as synaptogenesis and functional differentiation into type I and type II hair cells. In addition, we find that the developmental acquisition of basolateral conductances shapes the hair cell receptor potential and therefore comprises an important step in the signal cascade from mechanotransduction to neurotransmission.

Dominant-Negative Inhibition of M-Like Potassium Conductances in Hair Cells of the Mouse Inner Ear

Sensory hair cells of the inner ear express multiple physiologically defined conductances, including mechanotransduction, Ca2+, Na+, and several distinct K+ conductances, all of which are critical for normal hearing and balance function. Yet, the molecular underpinnings and their specific contributions to sensory signaling in the inner ear remain obscure. We sought to identify hair-cell conductances mediated by KCNQ4, which, when mutated, causes the dominant progressive hearing loss DFNA2. We used the dominant-negative pore mutation G285S and packaged the coding sequence of KCNQ4 into adenoviral vectors. We transfected auditory and vestibular hair cells of organotypic cultures generated from the postnatal mouse inner ear. Cochlear outer hair cells and vestibular type I cells that expressed the transfection marker, green fluorescent protein, and the dominant-negative KCNQ4 construct lacked the M-like conductances that typify nontransfected control hair cells. As such, we conclude that the M-like conductances in mouse auditory and vestibular hair cells can include KCNQ4 subunits and may also include KCNQ4 coassembly partners. To examine the function of M-like conductances in hair cells, we recorded from cells transfected with mutant KCNQ4 and injected transduction current waveforms in current-clamp mode. Because the M-like conductances were active at rest, they contributed to the very low potassium-selective input resistance, which in turn hyperpolarized the resting potential and significantly attenuated the amplitude of the receptor potential. Modulation of M-like conductances may allow hair cells the ability to control the amplitude of their response to sensory stimuli.