Cisplatin is a widely used anticancer drug with notable side effects including ototoxicity and nephrotoxicity. Macrophages, the major resident immune cells in the cochlea and kidney, are important drivers of both inflammatory and tissue repair responses. To investigate the roles of macrophages in cisplatin-induced toxicities, we used PLX3397, a U.S. Food and Drug Administration-approved inhibitor of the colony-stimulating factor 1 receptor, to eliminate tissue-resident macrophages. Mice treated with cisplatin alone had considerable hearing loss (ototoxicity) and kidney injury (nephrotoxicity). Macrophage ablation resulted in significantly reduced hearing loss and had greater outer hair cell survival. Macrophage ablation also protected against cisplatin-induced nephrotoxicity, as evidenced by markedly reduced tubular injury and fibrosis. Mechanistically, our data suggest that the protective effect of macrophage ablation against cisplatin-induced ototoxicity and nephrotoxicity is mediated by reduced platinum accumulation in both the inner ear and the kidney. Together, our data indicate that ablation of tissue-resident macrophages represents an important strategy for mitigating cisplatin-induced ototoxicity and nephrotoxicity.

Cisplatin is a chemotherapy drug that causes permanent hearing loss by injuring cochlear hair cells. The mechanisms that initiate injury are not fully understood, but mitochondria have emerged as potential mediators of hair cell cytotoxicity. Using in vivo live imaging of hair cells in the zebrafish lateral-line organ expressing a genetically encoded indicator of cumulative mitochondrial activity, we first demonstrate that greater redox history increases susceptibility to cisplatin. Next, we conducted time-lapse imaging to understand dynamic changes in mitochondrial homeostasis and observe elevated mitochondrial and cytosolic calcium that surge prior to hair cell death. Furthermore, using a localized probe that fluoresces in the presence of cisplatin, we show that cisplatin directly accumulates in hair cell mitochondria, and this accumulation occurs before mitochondrial dysregulation and apoptosis. Our findings provide evidence that cisplatin directly targets hair cell mitochondria and support that the mitochondria are integral to cisplatin cytotoxicity in hair cells.

Spiral ganglion neurons (SGNs) transmit sensory data from cochlear hair cells to the cochlear nuclei of the brainstem. Loss of these neurons deprives the brain of information on sound, so functioning SGNs are necessary both for normal hearing and for the restoration of hearing with cochlear prostheses. This chapter provides a brief introduction to SGNs and their peripheral and central connections. Then, we summarize three types of SGN pathology and possible methods for their repair, loss of synapses, loss of axons, and loss of neurons entirely. Afferent synapses on inner hair cells can be lost as a consequence of noise exposure or aging, and we discuss experimental approaches to promote synapse regeneration. Aging and noise can also cause death of SGNs, which may occur slowly and involve the degeneration of peripheral axons. We discuss possible approaches to maintaining or regenerating these axons, which may help improve the efficacy of cochlear implants. Finally, we discuss current attempts to use stem cells for the replacement of lost SGNs, along with some of the associated challenges.

Resident cochlear macrophages rapidly migrate into the inner hair cell synaptic region and directly contact the damaged synaptic connections after noise-induced synaptopathy. Eventually, such damaged synapses are spontaneously repaired, but the precise role of macrophages in synaptic degeneration and repair remains unknown. To address this, cochlear macrophages were eliminated using colony stimulating factor 1 receptor (CSF1R) inhibitor, PLX5622. Sustained treatment with PLX5622 in CX 3 CR1 GFP /+ mice of both sexes led to robust elimination of resident macrophages (∼94%) without significant adverse effects on peripheral leukocytes, cochlear function, and structure. At 1 day (d) post noise exposure of 93 or 90 dB SPL for 2 hours, the degree of hearing loss and synapse loss were comparable in the presence and absence of macrophages. At 30 d after exposure, damaged synapses appeared repaired in the presence of macrophages. However, in the absence of macrophages, such synaptic repair was significantly reduced. Remarkably, on cessation of PLX5622 treatment, macrophages repopulated the cochlea, leading to enhanced synaptic repair. Elevated auditory brainstem response thresholds and reduced auditory brainstem response Peak 1 amplitudes showed limited recovery in the absence of macrophages but recovered similarly with resident and repopulated macrophages. Cochlear neuron loss was augmented in the absence of macrophages but showed preservation with resident and repopulated macrophages after noise exposure. While the central auditory effects of PLX5622 treatment and microglia depletion remain to be investigated, these data demonstrate that macrophages do not affect synaptic degeneration but are necessary and sufficient to restore cochlear synapses and function after noise-induced synaptopathy. SIGNIFICANCE STATEMENT The synaptic connections between cochlear inner hair cells and spiral ganglion neurons can be lost because of noise over exposure or biological aging. This loss may represent the most common causes of sensorineural hearing loss also known as hidden hearing loss. Synaptic loss results in degradation of auditory information, leading to difficulty in listening in noisy environments and other auditory perceptual disorders. We demonstrate that resident macrophages of the cochlea are necessary and sufficient to restore synapses and function following synaptopathic noise exposure. Our work reveals a novel role for innate-immune cells, such as macrophages in synaptic repair, that could be harnessed to regenerate lost ribbon synapses in noise- or age-linked cochlear synaptopathy, hidden hearing loss, and associated perceptual anomalies.

Hair cells are the sensory receptors of the vertebrate inner ear that detect sound vibrations (to initiate hearing) and head movements (to facilitate balance). In humans, injury or loss of these cells can cause permanent hearing impairment, disequilibrium, and/or improper visual reflexes. For this reason, the development of methods to promote the regeneration of lost hair cells is a topic of great translational interest. The demonstration that the ears of nonmammalian vertebrates can regenerate hair cells after injury has led to considerable research focused on understanding the biological basis of this regenerative process. Other work has attempted to induce repair in the mammalian inner ear via gene therapy or introduction of stem cells. Such research is still ongoing, and it is not clear which approaches will ultimately yield clinical strategies for restoring hearing and motion perception. This chapter provides a brief history of this discipline and summarizes the contents of this volume.

Cisplatin is a commonly used chemotherapeutic agent that causes debilitating high-frequency hearing loss. No targeted therapies currently exist to treat cisplatin ototoxicity, partly because the underlying mechanisms of cisplatin-induced hair cell damage are not completely defined. Zebrafish may offer key insights to cisplatin ototoxicity because their lateral-line organ contains hair cells that are remarkably similar to those within the cochlea but are optically accessible, permitting observation of cisplatin injury in live intact hair cells. In this study, we used a combination of genetically encoded biosensors in zebrafish larvae and fluorescent indicators to characterize changes in mitochondrial bioenergetics in response to cisplatin. Following exposure to cisplatin, confocal imaging of live intact neuromasts demonstrated increased mitochondrial activity. Staining with fixable fluorescent dyes that accumulate in active mitochondria similarly showed hyperpolarized mitochondrial membrane potential. Zebrafish expressing a calcium indicator within their hair cells revealed elevated levels of mitochondrial calcium immediately following completion of cisplatin treatment. A fluorescent ROS indicator demonstrated that these changes in mitochondrial function were associated with increased oxidative stress. After a period of recovery, cisplatin-exposed zebrafish demonstrated caspase-3-mediated apoptosis. Altogether, these findings suggest that cisplatin acutely disrupts mitochondrial bioenergetics and may play a key role in initiating cisplatin ototoxicity.

Cisplatin is an effective anticancer agent, but also causes permanent hearing loss by damaging hair cells—the sensory receptors essential for hearing. There is an urgent clinical need to protect cochlear hair cells in patients undergoing cisplatin chemotherapy. The zebrafish lateral line organ contains hair cells and has been frequently used in studies to screen for otoprotective compounds. However, these studies have employed a wide range of cisplatin dosages and exposure times. We therefore performed a comprehensive evaluation of cisplatin ototoxicity in the zebrafish lateral line with the goal of producing a standardized, clinically relevant protocol for future studies. To define the dose- and time-response patterns of cisplatin-induced hair-cell death, we treated 6-day-old larvae for 2 h in 50 µM–1 mM cisplatin and allowed them to recover. We observed delayed hair cell death, which peaked at 4–8 h post-exposure. Cisplatin also activated a robust inflammatory response, as determined by macrophage recruitment and phagocytosis of hair cells. However, selective depletion of macrophages did not affect hair cell loss. We also examined the effect of cisplatin treatment on fish behavior and found that cisplatin-induced lateral line injury measurably impaired rheotaxis. Finally, we examined the function of remaining hair cells that appeared resistant to cisplatin treatment. We observed significantly reduced uptake of the cationic dye FM1-43 in these cells relative to untreated controls, indicating that surviving hair cells may be functionally impaired. Cumulatively, these results indicate that relatively brief exposures to cisplatin can produce hair cell damage and delayed hair cell death. Our observations provide guidance on standardizing methods for the use of the zebrafish model in studies of cisplatin ototoxicity.

Programmed cell death (PCD) plays a critical role in the development and maturation of the cochlea. Significant remodeling occurs among cells of the greater epithelial ridge (GER) of Kölliker’s organ, leading to tissue regression and formation of the inner sulcus. In mice, this event normally occurs between postnatal days 5–15 (P5-15) and is regulated by thyroid hormone (T3). During this developmental time period, the cochlea also contains a large population of macrophages. Macrophages are frequently involved in the phagocytic clearance of dead cells, both during development and after injury, but the role of macrophages in the developing cochlea is unknown. This study examined the link between developmental cell death in the GER and the recruitment of macrophages into this region. Cell death in the basal GER begins at P5 and enhanced numbers of macrophages were observed at P7. This pattern of macrophage recruitment was unchanged in mice that were genetically deficient for CX3CR1, the receptor for fractalkine (a known macrophage chemoattractant). We found that injection of T3 at P0 and P1 caused GER cell death to begin at P3, and this premature PCD was accompanied by earlier recruitment of macrophages. We further found that depletion of macrophages from the developing cochlea (using CX3CR1DTR/+ mice and treatment with the CSF1R antagonist BLZ945) had no effect on the pattern of GER regression. Together, these findings suggest that macrophages are recruited into the GER region after initiation of developmental PCD, but that they are not essential for GER regression during cochlear remodeling.

The sensory organs of the inner ear contain resident populations of macrophages, which are recruited to sites of cellular injury. Such macrophages are known to phagocytose the debris of dying cells but the full role of macrophages in otic pathology is not understood. Lateral line neuromasts of zebrafish contain hair cells that are nearly identical to those in the inner ear, and the optical clarity of larval zebrafish permits direct imaging of cellular interactions. In this study, we used larval zebrafish to characterize the response of macrophages to ototoxic injury of lateral line hair cells. Macrophages migrated into neuromasts within 20 min of exposure to the ototoxic antibiotic neomycin. The number of macrophages in the near vicinity of injured neuromasts was similar to that observed near uninjured neuromasts, suggesting that this early inflammatory response was mediated by "local" macrophages. Upon entering injured neuromasts, macrophages actively phagocytosed hair cell debris. The injury-evoked migration of macrophages was significantly reduced by inhibition of Src-family kinases. Using chemical-genetic ablation of macrophages before the ototoxic injury, we also examined whether macrophages were essential for the initiation of hair cell regeneration. Results revealed only minor differences in hair cell recovery in macrophage-depleted vs. control fish, suggesting that macrophages are not essential for the regeneration of lateral line hair cells.

The Hippo signaling pathway is a key regulator of tissue development and regeneration. Activation of the Hippo pathway leads to nuclear translocation of the YAP1 transcriptional coactivator, resulting in changes in gene expression and cell cycle entry. Recent studies have demonstrated the nuclear translocation of YAP1 during the development of the sensory organs of the inner ear, but the possible role of YAP1 in sensory regeneration of the inner ear is unclear. The present study characterized the cellular localization of YAP1 in the utricles of mice and chicks, both under normal conditions and after HC injury. During neonatal development, YAP1 expression was observed in the cytoplasm of supporting cells, and was transiently expressed in the cytoplasm of some differentiating hair cells. We also observed temporary nuclear translocation of YAP1 in supporting cells of the mouse utricle after short periods in organotypic culture. However, little or no nuclear translocation of YAP1 was observed in the utricles of neonatal or mature mice after ototoxic injury. In contrast, substantial YAP1 nuclear translocation was observed in the chicken utricle after streptomycin treatment in vitro and in vivo. Together, these data suggest that differences in YAP1 signaling may partially account for the differing regenerative abilities of the avian vs. mammalian inner ear.

Excess noise damages sensory hair cells, resulting in loss of synaptic connections with auditory nerves and, in some cases, hair-cell death. The cellular mechanisms underlying mechanically induced hair-cell damage and subsequent repair are not completely understood. Hair cells in neuromasts of larval zebrafish are structurally and functionally comparable to mammalian hair cells but undergo robust regeneration following ototoxic damage. We therefore developed a model for mechanically induced hair-cell damage in this highly tractable system. Free swimming larvae exposed to strong water wave stimulus for 2 hr displayed mechanical injury to neuromasts, including afferent neurite retraction, damaged hair bundles, and reduced mechanotransduction. Synapse loss was observed in apparently intact exposed neuromasts, and this loss was exacerbated by inhibiting glutamate uptake. Mechanical damage also elicited an inflammatory response and macrophage recruitment. Remarkably, neuromast hair-cell morphology and mechanotransduction recovered within hours following exposure, suggesting severely damaged neuromasts undergo repair. Our results indicate functional changes and synapse loss in mechanically damaged lateral-line neuromasts that share key features of damage observed in noise-exposed mammalian ear. Yet, unlike the mammalian ear, mechanical damage to neuromasts is rapidly reversible.

Hair cells in the auditory organ of the vertebrate inner ear are the sensory receptors that convert acoustic stimuli into electrical signals that are conveyed along the auditory nerve to the brainstem. Hair cells are highly susceptible to ototoxic drugs, infection, and acoustic trauma, which can cause cellular degeneration. In mammals, hair cells that are lost after damage are not replaced, leading to permanent hearing impairments. By contrast, supporting cells in birds and other non-mammalian vertebrates regenerate hair cells after damage, which restores hearing function. The cellular mechanisms that regulate hair cell regeneration are not well understood. We investigated the role of vascular endothelial growth factor (VEGF) during regeneration of auditory hair cells in chickens after ototoxic injury. Using RNA-Seq, immunolabeling, and in situ hybridization, we found that VEGFA, VEGFC, VEGFR1, VEGFR2, and VEGFR3 were expressed in the auditory epithelium, with VEGFA expressed in hair cells and VEGFR1 and VEGFR2 expressed in supporting cells. Using organotypic cultures of the chicken cochlear duct, we found that blocking VEGF receptor activity during hair cell injury reduced supporting cell proliferation as well as the numbers of regenerated hair cells. By contrast, addition of recombinant human VEGFA to organ cultures caused an increase in both supporting cell division and hair cell regeneration. VEGF's effects on supporting cells were preserved in isolated supporting cell cultures, indicating that VEGF can act directly upon supporting cells. These observations demonstrate a heretofore uncharacterized function for VEGF signaling as a critical positive regulator of hair cell regeneration in the avian inner ear.

Precise control of organ growth and patterning is executed through a balanced regulation of progenitor self-renewal and differentiation. In the auditory sensory epithelium-the organ of Corti-progenitor cells exit the cell cycle in a coordinated wave between E12.5 and E14.5 before the initiation of sensory receptor cell differentiation, making it a unique system for studying the molecular mechanisms controlling the switch between proliferation and differentiation. Here we identify the Yap/Tead complex as a key regulator of the self-renewal gene network in organ of Corti progenitor cells. We show that Tead transcription factors bind directly to the putative regulatory elements of many stemness- and cell cycle-related genes. We also show that the Tead coactivator protein, Yap, is degraded specifically in the Sox2-positive domain of the cochlear duct, resulting in down-regulation of Tead gene targets. Further, conditional loss of the Yap gene in the inner ear results in the formation of significantly smaller auditory and vestibular sensory epithelia, while conditional overexpression of a constitutively active version of Yap, Yap5SA, is sufficient to prevent cell cycle exit and to prolong sensory tissue growth. We also show that viral gene delivery of Yap5SA in the postnatal inner ear sensory epithelia in vivo drives cell cycle reentry after hair cell loss. Taken together, these data highlight the key role of the Yap/Tead transcription factor complex in maintaining inner ear progenitors during development, and suggest new strategies to induce sensory cell regeneration.

Noise trauma causes loss of synaptic connections between cochlear inner hair cells (IHCs) and the spiral ganglion neurons (SGNs). Such synaptic loss can trigger slow and progressive degeneration of SGNs. Macrophage fractalkine signaling is critical for neuron survival in the injured cochlea, but its role in cochlear synaptopathy is unknown. Fractalkine, a chemokine, is constitutively expressed by SGNs and signals via its receptor CX(3)CR1 that is expressed on macrophages. The present study characterized the immune response and examined the function of fractalkine signaling in degeneration and repair of cochlear synapses following noise trauma. Adult mice wild type, heterozygous and knockout for CX(3)CR1 on a C57BL/6 background were exposed for 2 h to an octave band noise at 90 dB SPL. Noise exposure caused temporary shifts in hearing thresholds without any evident loss of hair cells in CX(3)CR1 heterozygous mice that have intact fractalkine signaling. Enhanced macrophage migration toward the IHC-synaptic region was observed immediately after exposure in all genotypes. Synaptic immunolabeling revealed a rapid loss of ribbon synapses throughout the basal turn of the cochlea of all genotypes. The damaged synapses spontaneously recovered in mice with intact CX(3)CR1. However, CX(3)CR1 knockout (KO) animals displayed enhanced synaptic degeneration that correlated with attenuated suprathreshold neural responses at higher frequencies. Exposed CX(3)CR1 KO mice also exhibited increased loss of IHCs and SGN cell bodies compared to exposed heterozygous mice. These results indicate that macrophages can promote repair of damaged synapses after moderate noise trauma and that repair requires fractalkine signaling.

Macrophages are present in most somatic tissues, where they detect and attack invading pathogens. Macrophages also participate in many nonimmune functions, particularly those related to tissue maintenance and injury response. The sensory organs of the inner ear contain resident populations of macrophages, and additional macrophages enter the ear after acoustic trauma orototoxicity. As expected, such macrophages participate in the clearance of cellular debris. However, otic macrophages can also influence the long-term survival of both hair cells and afferent neurons after injury. The signals that recruit macrophages into the injured ear, as well as the precise contributions of macrophages to inner ear pathology, remain to be determined.

The vestibular organs of reptiles, birds, and mammals possess Type I and Type II sensory hair cells, which have distinct morphologies, physiology, and innervation. Little is known about how vestibular hair cells adopt a Type I or Type II identity or acquire proper innervation. One distinguishing marker is the transcription factor Sox2, which is expressed in all developing hair cells but persists only in Type II hair cells in maturity. We examined Sox2 expression and formation of afferent nerve terminals in mouse utricles between postnatal days 0 (P0) and P17. Between P3 and P14, many hair cells lost Sox2 immunoreactivity and the density of calyceal afferent nerve terminals (specific to Type I hair cells) increased in all regions of the utricle. At early time points, many calyces enclosed Sox2-labeled hair cells, while some Sox2-negative hair cells within the striola had not yet developed a calyx. These observations indicate that calyx maturation is not temporally correlated with loss of Sox2 expression in Type I hair cells. To determine which type(s) of hair cells are formed postnatally, we fate-mapped neonatal supporting cells by injecting Plp-CreER(T2):Rosa26(tdTomato) mice with tamoxifen at P2 and P3. At P9, tdTomato-positive hair cells were immature and not classifiable by type. At P30, tdTomato-positive hair cells increased 1.8-fold compared to P9, and 91% of tdTomato-labeled hair cells were Type II. Our findings show that most neonatally-derived hair cells become Type II, and many Type I hair cells (formed before P2) downregulate Sox2 and acquire calyces between P0 and P14.

The endocochlear potential (EP) provides part of the electrochemical drive for sound-driven currents through cochlear hair cells. Intense noise exposure (110 dB SPL, 2 h) differentially affects the EP in three inbred mouse strains (C57BL/6 [136], CBA/J [CBA], BALB/cJ [BALM (Ohlemiller and Gagnon, 2007, Hearing Research 224:34-50; Ohlemiller et al., 2011, JARO 12:45-58). At least for mice older than 3 mos, B6 mice are unaffected, CBA mice show temporary EP reduction, and BALB mice may show temporary or permanent EP reduction. EP reduction was well correlated with histological metrics for injury to stria vascularis and spiral ligament, and little evidence was found for holes or tears in the reticular lamina that might 'short out' the EP. Thus we suggested that the genes and processes that underlie the strain EP differences primarily impact cochlear lateral wall, not the organ of Corti. Our previous work did not test the range of noise exposure conditions over which strain differences apply. It therefore remained possible that the relation between exposure severity and acute EP reduction simply has a higher exposure threshold in B6 mice compared to CBA and BALB. We also did not test for age dependence. It is well established that young adult animals are especially vulnerable to noise-induced permanent threshold shifts (NIPTS). It is unknown, however, whether heightened vulnerability of the lateral wall contributes to this condition. The present study extends our previous work to multiple noise exposure levels and durations, and explicitly compares young adult (6-7 wks) and older mice (>4 mos). We find that the exposure level-versus-acute EP relation is dramatically strain-dependent, such that 136 mice widely diverge from both CBA and BALB. For all three strains, however, acute EP reduction is greater in young mice. Above 110 dB SPL, all mice exhibited rapid and severe EP reduction that is likely related to tearing of the reticular lamina. By contrast, EP-versus-noise duration examined at 104 dB suggested that different processes contribute to EP reduction in young and older mice. The average EP falls to a constant level after similar to 7.5 min in older mice, but progressively decreases with further exposure in young mice. Confocal microscopy of organ of Corti surface preparations stained for phalloidin and zonula occludens-1 (ZO-1) indicated this corresponds to rapid loss of outer hair cells (OHCs) and formation of both holes and tears in the reticular lamina of young mice. In addition, when animals exposed at 119 dB were allowed to recover for 1 mo, only young B6 mice showed collapse of the EP to <5 mV. Confocal analysis suggested novel persistent loss of tight junctions in the lateral organ of Corti. This may allow paracellular leakage that permanently reduces the EP. From our other findings, we propose that noise-related lateral wall pathology in young CBA and BALB mice promotes hair cell loss and opening of the reticular lamina. The heightened vulnerability of young adult animals to noise exposure may in part reflect special sensitivity of the organ of Corti to acute lateral wall dysfunction at younger ages. This feature appears genetically modifiable. (C) 2018 Elsevier B.V. All rights reserved.

Cochlear hair cells are vulnerable to a variety of insults like acoustic trauma and ototoxic drugs. Such injury can also lead to degeneration of spiral ganglion neurons (SGNs), but this occurs over a period of months to years. Neuronal survival is necessary for the proper function of cochlear prosthetics, therefore, it is of great interest to understand the mechanisms that regulate neuronal survival in deaf ears. We have recently demonstrated that selective hair cell ablation is sufficient to attract leukocytes into the spiral ganglion, and that fractalkine signaling plays a role in macrophage recruitment and in the survival of auditory neurons. Fractalkine (CX 3 CL1), a chemokine that regulates adhesion and migration of leukocytes is expressed by SGNs and signals to leukocytes via its receptor CX 3 CR1. The present study has extended the previous findings to more clinically relevant conditions of sensorineural hearing loss by examining the role of fractalkine signaling after aminoglycoside ototoxicity or acoustic trauma. Both aminoglycoside treatment and acoustic overstimulation led to the loss of hair cells as well as prolonged increase in the numbers of cochlear leukocytes. Lack of CX 3 CR1 did not affect macrophage recruitment after injury, but resulted in increased loss of SGNs and enhanced expression of the inflammatory cytokine interleukin‐1β, when compared to mice with intact CX 3 CR1. These data indicate that the dysregulation of macrophage response caused by the absence of CX 3 CR1 may contribute to inflammation‐mediated neuronal loss in the deafened ear, suggesting a key role for inflammation in the long‐term survival of target‐deprived afferent neurons.

The loss of sensory hair cells from the inner ear is a leading cause of hearing and balance disorders. The mammalian ear has a very limited ability to replace lost hair cells, but the inner ears of non-mammalian vertebrates can spontaneously regenerate hair cells after injury. Prior studies have shown that replacement hair cells are derived from epithelial supporting cells and that the differentiation of new hair cells is regulated by the Notch signaling pathway. The present study examined molecular influences on regeneration in the avian utricle, which has a particularly robust regenerative ability. Chicken utricles were placed in organotypic culture and hair cells were lesioned by application of the ototoxic antibiotic streptomycin. Cultures were then allowed to regenerate in vitro for seven days. Some specimens were treated with small molecule inhibitors of γ-secretase or ADAM10, proteases which are essential for transmission of Notch signaling. As expected, treatment with both inhibitors led to increased numbers of replacement hair cells. However, we also found that inhibition of both proteases resulted in increased regenerative proliferation. Subsequent experiments showed that inhibition of γ-secretase or ADAM10 could also trigger proliferation in undamaged utricles. To better understand these phenomena, we used RNA-Seq profiling to characterize changes in gene expression following γ-secretase inhibition. We observed expression patterns that were consistent with Notch pathway inhibition, but we also found that the utricular sensory epithelium contains numerous γ-secretase substrates that might regulate cell cycle entry and possibly supporting cell-to-hair cell conversion. Together, our data suggest multiple roles for γ-secretase and ADAM10 in vestibular hair cell regeneration.