PKHD1L1 is required for stereocilia bundle maintenance, durable hearing function and resilience to noise exposure.

Polycystic Kidney and Hepatic Disease 1-Like 1 (PKHD1L1) is a human deafness gene, responsible for autosomal recessive deafness-124 (DFNB124). Sensory hair cells of the cochlea are essential for hearing, relying on the mechanosensitive stereocilia bundle at their apical pole for their function. PKHD1L1 is a stereocilia protein required for the formation of the developmentally transient stereocilia surface coat. In this study, we carry out an in depth characterization of PKHD1L1 expression in mice during development and adulthood, analyze hair-cell bundle morphology and hearing function in aging PKHD1L1-deficient mouse lines, and assess their susceptibility to noise damage. Our findings reveal that PKHD1L1-deficient mice display no disruption to bundle cohesion or tectorial membrane attachment-crown formation during development. However, starting from 6 weeks of age, PKHD1L1-deficient mice display missing stereocilia and disruptions to bundle coherence. Both conditional and constitutive PKHD1L1 knockout mice develop high-frequency hearing loss progressing to lower frequencies with age. Furthermore, PKHD1L1-deficient mice are susceptible to permanent hearing loss following moderate acoustic overexposure, which induces only temporary hearing threshold shifts in wild-type mice. These results suggest a role for PKHD1L1 in establishing robust sensory hair bundles during development, necessary for maintaining bundle cohesion and function in response to acoustic trauma and aging.

The Critical Thing about the Ear's Sensory Hair Cells.

The capabilities of the human ear are remarkable. We can normally detect acoustic stimuli down to a threshold sound-pressure level of 0 dB (decibels) at the entrance to the external ear, which elicits eardrum vibrations in the picometer range. From this threshold up to the onset of pain, 120 dB, our ears can encompass sounds that differ in power by a trillionfold. The comprehension of speech and enjoyment of music result from our ability to distinguish between tones that differ in frequency by only 0.2%. All these capabilities vanish upon damage to the ear's receptors, the mechanoreceptive sensory hair cells. Each cochlea, the auditory organ of the inner ear, contains some 16,000 such cells that are frequency-tuned between ∼20 Hz (cycles per second) and 20,000 Hz. Remarkably enough, hair cells do not simply capture sound energy: they can also exhibit an active process whereby sound signals are amplified, tuned, and scaled. This article describes the active process in detail and offers evidence that its striking features emerge from the operation of hair cells on the brink of an oscillatory instability-one example of the critical phenomena that are widespread in physics.

foxg1a is required for hair cell development and regeneration in the zebrafish lateral line.

Mechanosensory hair cells located in the inner ear mediate the sensations of hearing and balance. If damaged, mammalian inner ear hair cells are unable to regenerate, resulting in permanent sensory deficits. Aquatic vertebrates like zebrafish (Danio rerio) have a specialized class of mechanosensory hair cells found in the lateral line system, allowing them to sense changes in water current. Unlike mammalian inner ear hair cells, lateral line hair cells can robustly regenerate following damage. In mammals, the transcription factor Foxg1 functions to promote normal development of the inner ear. Foxg1a is expressed in lateral line sensory organs in zebrafish larvae, but its function during lateral line development and regeneration has not been investigated. Our study demonstrates that mutation of foxg1a results in slower posterior lateral line primordium migration and delayed neuromast formation. In developing and regenerating neuromasts, we find that loss of Foxg1a function results in reduced hair cell numbers, as well as decreased proliferation of neuromast cells. Foxg1a specifically regulates the development and regeneration of Islet1-labeled hair cells. These data suggest that Foxg1 may be a valuable target for investigation of clinical hair cell regeneration.

Neural control and innate self-tuning of the hair cell's active process.

We propose a model for the feedback control processes that underlie the robustness and high sensitivity of mechanosensory hair cells. Our model encompasses self-tuning active processes intrinsic to these cells, which drive the amplification of mechanical stimuli by consuming metabolic energy, and a neural input process that protects these cells from damage caused by powerful stimuli. We explore the effects of these two feedback mechanisms on mechanical self-oscillations of the sense cells and their response to external forcing.

Proteins required for stereocilia elongation during mammalian hair cell development ensure precise and steady heights during adult life.

The hair bundle, or stereocilia bundle, is the mechanosensory compartment of hair cells (HCs) in the inner ear. To date, most mechanistic studies have focused on stereocilia bundle morphogenesis, and it remains unclear how this organelle critical for hearing preserves its precise dimensions during life in mammals. The GPSM2-GNAI complex occupies the distal tip of stereocilia in the tallest row and is required for their elongation during development. Here, we ablate GPSM2-GNAI in adult mouse HCs after normal stereocilia elongation is completed. We observe a progressive height reduction of the tallest row stereocilia totaling ~600 nm after 12 wk in Gpsm2 mutant inner HCs. To measure GPSM2 longevity at tips, we generated a HaloTag-Gpsm2 mouse strain and performed pulse-chase experiments in vivo. Estimates using pulse-chase or tracking loss of GPSM2 immunolabeling following Gpsm2 inactivation suggest that GPSM2 is relatively long-lived at stereocilia tips with a half-life of 9 to 10 d. Height reduction coincides with dampened auditory brainstem responses evoked by low-frequency stimuli in particular. Finally, GPSM2 is required for normal tip enrichment of elongation complex (EC) partners MYO15A, WHRN, and EPS8, mirroring their established codependence during development. Taken together, our results show that the EC is also essential in mature HCs to ensure precise and stable stereocilia height and for sensitive detection of a full range of sound frequencies.

LOXHD1 is indispensable for maintaining TMC1 auditory mechanosensitive channels at the site of force transmission.

Hair cell bundles consist of stereocilia arranged in rows of increasing heights, connected by tip links that transmit sound-induced forces to shorter stereocilia tips. Auditory mechanotransduction channel complexes, composed of proteins TMC1/2, TMIE, CIB2, and LHFPL5, are located at the tips of shorter stereocilia. While most components can interact with the tip link in vitro, their ability to maintain the channel complexes at the tip link in vivo is uncertain. Return, using mouse models, we show that an additional component, LOXHD1, is essential for keeping TMC1-pore forming subunits at the tip link but is dispensable for TMC2. Using SUB-immunogold-SEM, we showed that TMC1 localizes near the tip link but mislocalizes without LOXHD1. LOXHD1 selectively interacts with TMC1, CIB2, LHFPL5, and tip-link protein PCDH15. Our results demonstrate that TMC1-driven mature auditory channels require LOXHD1 to stay connected to the tip link and remain functional, while TMC2-driven developmental channels do not.

The role of cilia in the development, survival, and regeneration of hair cells.

Mutations impacting cilia genes lead to a class of human diseases known as ciliopathies. This is due to the role of cilia in the development, survival, and regeneration of many cell types. We investigated the extent to which disrupting cilia impacted these processes in lateral line hair cells of zebrafish. We found that mutations in two intraflagellar transport (IFT) genes, ift88 and dync2h1, which lead to the loss of kinocilia, caused increased hair cell apoptosis. IFT gene mutants also have a decreased mitochondrial membrane potential, and blocking the mitochondrial uniporter causes a loss of hair cells in wild-type zebrafish but not mutants, suggesting mitochondria dysfunction may contribute to the apoptosis seen in these mutants. These mutants also showed decreased proliferation during hair cell regeneration but did not show consistent changes in support cell number or proliferation during hair cell development. These results show that the loss of hair cells seen following disruption of cilia through either mutations in anterograde or retrograde IFT genes appears to be due to impacts on hair cell survival but not necessarily development in the zebrafish lateral line.

3D Computational Modeling of Blast Wave Transmission in Human Ear From External Ear to Cochlear Hair Cells: A Preliminary Study.

INTRODUCTION: Auditory disabilities like tinnitus and hearing loss caused by exposure to blast overpressures are prevalent among military service members and veterans. The high-pressure fluctuations of blast waves induce hearing loss by injuring the tympanic membrane, ossicular chain, or sensory hair cells in the cochlea. The basilar membrane (BM) and organ of Corti (OC) behavior inside the cochlea during blast remain understudied. A computational finite element (FE) model of the full human ear was used by Bradshaw et al. (2023) to predict the motion of middle and inner ear tissues during blast exposure using a 3-chambered cochlea with Reissner's membrane and the BM. The inclusion of the OC in a blast transmission model would improve the model's anatomy and provide valuable insight into the inner ear response to blast exposure. MATERIALS AND METHODS: This study developed a microscale FE model of the OC, including the OC sensory hair cells, membranes, and structural cells, connected to a macroscale model of the ear to form a comprehensive multiscale model of the human peripheral auditory system. There are 5 rows of hair cells in the model, each row containing 3 outer hair cells (OHCs) and the corresponding Deiters' cells and stereociliary hair bundles. BM displacement 16.75 mm from the base induced by a 31 kPa blast overpressure waveform was derived from the macroscale human ear model reported by Bradshaw et al. (2023) and applied as input to the center of the BM in the OC. The simulation was run for 2 ms as a structural analysis in ANSYS Mechanical. RESULTS: The FE model results reported the displacement and principal strain of the OHCs, reticular lamina, and stereociliary hair bundles during blast transmission. The movement of the BM caused the rest of the OC to deform significantly. The reticular lamina displacement and strain amplitudes were highest where it connected to the OHCs, indicating that injury to this part of the OC may be likely due to blast exposure. CONCLUSIONS: This microscale model is the first FE model of the OC to be connected to a macroscale model of the ear, forming a full multiscale ear model, and used to predict the OC's behavior under blast. Future work with this model will incorporate cochlear endolymphatic fluid, increase the number of OHC rows to 19 in total, and use the results of the model to reliably predict the sensorineural hearing loss resulting from blast exposure.

The single-cell transcriptomic landscape of the topological differences in mammalian auditory receptors.

Mammalian hair cells (HCs) are arranged spirally along the cochlear axis and correspond to different frequency ranges. Serving as primary sound detectors, HCs spatially segregate component frequencies into a topographical map. HCs display significant diversity in anatomical and physiological characteristics, yet little is known about the organization of the cochleotopic map of HCs or the molecules involved in this process. Using single-cell RNA sequencing, we determined the distinct molecular profiles of inner hair cells and outer hair cells, and we identified numerous position-dependent genes that were expressed as gradients. Newly identified genes such as Ptn, Rxra, and Nfe2l2 were found to be associated with tonotopy. We employed the SCENIC algorithm to predict the transcription factors that potentially shape these tonotopic gradients. Furthermore, we confirmed that Nfe2l2, a tonotopy-related transcription factor, is critical in mice for sensing low-to-medium sound frequencies in vivo. the analysis of cell-cell communication revealed potential receptor-ligand networks linking inner hair cells to spiral ganglion neurons, including pathways such as BDNF-Ntrk and PTN-Scd4, which likely play essential roles in tonotopic maintenance. Overall, these findings suggest that molecular gradients serve as the organizing principle for maintaining the selection of sound frequencies by HCs.

Auditory hair cells and spiral ganglion neurons regenerate synapses with refined release properties in vitro.

Ribbon synapses between inner hair cells (IHCs) and type I spiral ganglion neurons (SGNs) in the inner ear are damaged by noise trauma and with aging, causing "synaptopathy" and hearing loss. Cocultures of neonatal denervated organs of Corti and newly introduced SGNs have been developed to find strategies for improving IHC synapse regeneration, but evidence of the physiological normality of regenerated synapses is missing. This study utilizes IHC optogenetic stimulation and SGN recordings, showing that, when P3-5 denervated organs of Corti are cocultured with SGNs, newly formed IHC/SGN synapses are indeed functional, exhibiting glutamatergic excitatory postsynaptic currents. When using older organs of Corti at P10-11, synaptic activity probed by deconvolution showed more mature release properties, closer to the specialized mode of IHC synaptic transmission crucial for coding the sound signal. This functional assessment of newly formed IHC synapses developed here, provides a powerful tool for testing approaches to improve synapse regeneration.

Transdifferentiation is temporally uncoupled from progenitor pool expansion during hair cell regeneration in the zebrafish inner ear.

Death of mechanosensory hair cells in the inner ear is a common cause of auditory and vestibular impairment in mammals, which have a limited ability to regrow these cells after damage. In contrast, non-mammalian vertebrates, including zebrafish, can robustly regenerate hair cells after severe organ damage. The zebrafish inner ear provides an understudied model system for understanding hair cell regeneration in organs that are highly conserved with their mammalian counterparts. Here, we quantitatively examine hair cell addition during growth and regeneration of the larval zebrafish inner ear. We used a genetically encoded ablation method to induce hair cell death and we observed gradual regeneration with correct spatial patterning over a 2-week period following ablation. Supporting cells, which surround and are a source of new hair cells, divide in response to hair cell ablation, expanding the possible progenitor pool. In parallel, nascent hair cells arise from direct transdifferentiation of progenitor pool cells temporally uncoupled from supporting cell division. These findings reveal a previously unrecognized mechanism of hair cell regeneration with implications for how hair cells may be encouraged to regenerate in the mammalian ear.

Comparative biology of the amniote vestibular utricle.

The sensory epithelia of the auditory and vestibular systems of vertebrates have shared developmental and evolutionary histories. However, while the auditory epithelia show great variation across vertebrates, the vestibular sensory epithelia appear seemingly more conserved. An exploration of the current knowledge of the comparative biology of the amniote utricle, a vestibular sensory epithelium that senses linear acceleration, shows interesting instances of variability between birds and mammals. The distribution of sensory hair cell types, the position of the line of hair bundle polarity reversal and the properties of supporting cells show marked differences, likely impacting vestibular function and hair cell regeneration potential.

Priming central sound processing circuits through induction of spontaneous activity in the cochlea before hearing onset.

Sensory systems experience a period of intrinsically generated neural activity before maturation is complete and sensory transduction occurs. Here we review evidence describing the mechanisms and functions of this 'spontaneous' activity in the auditory system. Both ex vivo and in vivo studies indicate that this correlated activity is initiated by non-sensory supporting cells within the developing cochlea, which induce depolarization and burst firing of groups of nearby hair cells in the sensory epithelium, activity that is conveyed to auditory neurons that will later process similar sound features. This stereotyped neural burst firing promotes cellular maturation, synaptic refinement, acoustic sensitivity, and establishment of sound-responsive domains in the brain. While sensitive to perturbation, the developing auditory system exhibits remarkable homeostatic mechanisms to preserve periodic burst firing in deaf mice. Preservation of this early spontaneous activity in the context of deafness may enhance the efficacy of later interventions to restore hearing.

Conditions Underlying the Appearance of Spontaneous Otoacoustic Emissions in Mammals.

Across the wide range of land vertebrate species, spontaneous otoacoustic emissions (SOAE) are common, but not always found. The reasons for the differences between species of the various groups in their emission patterns are often not well understood, particularly within mammals. This review examines the question as to what determines in mammals whether SOAE are emitted or not, and suggests that the coupling between hair-cell regions diminishes when the space constant of frequency distribution becomes larger. The reduced coupling is assumed to result in a greater likelihood of SOAE being emitted.

Inhibitory G proteins play multiple roles to polarize sensory hair cell morphogenesis.

Inhibitory G alpha (GNAI or Gαi) proteins are critical for the polarized morphogenesis of sensory hair cells and for hearing. The extent and nature of their actual contributions remains unclear, however, as previous studies did not investigate all GNAI proteins and included non-physiological approaches. Pertussis toxin can downregulate functionally redundant GNAI1, GNAI2, GNAI3, and GNAO proteins, but may also induce unrelated defects. Here, we directly and systematically determine the role(s) of each individual GNAI protein in mouse auditory hair cells. GNAI2 and GNAI3 are similarly polarized at the hair cell apex with their binding partner G protein signaling modulator 2 (GPSM2), whereas GNAI1 and GNAO are not detected. In Gnai3 mutants, GNAI2 progressively fails to fully occupy the sub-cellular compartments where GNAI3 is missing. In contrast, GNAI3 can fully compensate for the loss of GNAI2 and is essential for hair bundle morphogenesis and auditory function. Simultaneous inactivation of Gnai2 and Gnai3 recapitulates for the first time two distinct types of defects only observed so far with pertussis toxin: (1) a delay or failure of the basal body to migrate off-center in prospective hair cells, and (2) a reversal in the orientation of some hair cell types. We conclude that GNAI proteins are critical for hair cells to break planar symmetry and to orient properly before GNAI2/3 regulate hair bundle morphogenesis with GPSM2.

Disruption of Cdh23 exon 68 splicing leads to progressive hearing loss in mice by affecting tip-link stability.

Inner ear hair cells are characterized by the F-actin-based stereocilia that are arranged into a staircase-like pattern on the apical surface of each hair cell. The tips of shorter-row stereocilia are connected with the shafts of their neighboring taller-row stereocilia through extracellular links named tip links, which gate mechano-electrical transduction (MET) channels in hair cells. Cadherin 23 (CDH23) forms the upper part of tip links, and its cytoplasmic tail is inserted into the so-called upper tip-link density (UTLD) that contains other proteins such as harmonin. The Cdh23 gene is composed of 69 exons, and we show here that exon 68 is subjected to hair cell-specific alternative splicing. Tip-link formation is not affected in genetically modified mutant mice lacking Cdh23 exon 68. Instead, the stability of tip links is compromised in the mutants, which also suffer from progressive and noise-induced hearing loss. Moreover, we show that the cytoplasmic tail of CDH23(+68) but not CDH23(-68) cooperates with harmonin in phase separation-mediated condensate formation. In conclusion, our work provides evidence that inclusion of Cdh23 exon 68 is critical for the stability of tip links through regulating condensate formation of UTLD components.

Hair cell regeneration, reinnervation, and restoration of hearing thresholds in the avian hearing organ.

Hearing starts, at the cellular level, with mechanoelectrical transduction by sensory hair cells. Sound information is then transmitted via afferent synaptic connections with auditory neurons. Frequency information is encoded by the location of hair cells along the cochlear duct. Loss of hair cells, synapses, or auditory neurons leads to permanent hearing loss in mammals. Birds, in contrast, regenerate auditory hair cells and functionally recover from hearing loss. Here, we characterized regeneration and reinnervation in sisomicin-deafened chickens and found that afferent neurons contact regenerated hair cells at the tips of basal projections. In contrast to development, synaptic specializations are established at these locations distant from the hair cells' bodies. The protrusions then contracted as regenerated hair cells matured and became functional 2 weeks post-deafening. We found that auditory thresholds recovered after 4-5 weeks. We interpret the regeneration-specific synaptic reestablishment as a location-preserving process that might be needed to maintain tonotopic fidelity.

Spherical harmonics analysis reveals cell shape-fate relationships in zebrafish lateral line neuromasts.

Cell shape is a powerful readout of cell state, fate and function. We describe a custom workflow to perform semi-automated, 3D cell and nucleus segmentation, and spherical harmonics and principal components analysis to distill cell and nuclear shape variation into discrete biologically meaningful parameters. We apply these methods to analyze shape in the neuromast cells of the zebrafish lateral line system, finding that shapes vary with cell location and identity. The distinction between hair cells and support cells accounted for much of the variation, which allowed us to train classifiers to predict cell identity from shape features. Using transgenic markers for support cell subpopulations, we found that subtypes had different shapes from each other. To investigate how loss of a neuromast cell type altered cell shape distributions, we examined atoh1a mutants that lack hair cells. We found that mutant neuromasts lacked the cell shape phenotype associated with hair cells, but did not exhibit a mutant-specific cell shape. Our results demonstrate the utility of using 3D cell shape features to characterize, compare and classify cells in a living developing organism.