Sensory Hair Cells Research Articles

A Novel Small Molecule Neurotrophin-3 Analogue Promotes Inner Ear Neurite Outgrowth and Synaptogenesis In vitro.

Sensorineural hearing loss is irreversible and is associated with the loss of spiral ganglion neurons (SGNs) and sensory hair cells within the inner ear. Improving spiral ganglion neuron (SGN) survival, neurite outgrowth, and synaptogenesis could lead to significant gains for hearing-impaired patients. There has therefore been intense interest in the use of neurotrophic factors in the inner ear to promote both survival of SGNs and re-wiring of sensory hair cells by surviving SGNs. Neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) represent the primary neurotrophins in the inner ear during development and throughout adulthood, and have demonstrated potential for SGN survival and neurite outgrowth. We have pioneered a hybrid molecule approach to maximize SGN stimulation in vivo, in which small molecule analogues of neurotrophins are linked to bisphosphonates, which in turn bind to cochlear bone. We have previously shown that a small molecule BDNF analogue coupled to risedronate binds to bone matrix and promotes SGN neurite outgrowth and synaptogenesis in vitro. Because NT-3 has been shown in a variety of contexts to have a greater regenerative capacity in the cochlea than BDNF, we sought to develop a similar approach for NT-3. 1Aa is a small molecule analogue of NT-3 that has been shown to activate cells through TrkC, the NT-3 receptor, although its activity on SGNs has not previously been described. Herein we describe the design and synthesis of 1Aa and a covalent conjugate of 1Aa with risedronate, Ris-1Aa. We demonstrate that both 1Aa and Ris-1Aa stimulate neurite outgrowth in SGN cultures at a significantly higher level compared to controls. Ris-1Aa maintained its neurotrophic activity when bound to hydroxyapatite, the primary mineral component of bone. Both 1Aa and Ris-1Aa promote significant synaptic regeneration in cochlear explant cultures, and both 1Aa and Ris-1Aa appear to act at least partly through TrkC. Our results provide the first evidence that a small molecule analogue of NT-3 can stimulate SGNs and promote regeneration of synapses between SGNs and inner hair cells. Our findings support the promise of hydroxyapatite-targeting bisphosphonate conjugation as a novel strategy to deliver neurotrophic agents to SGNs encased within cochlear bone.

Multiple PDZ domain protein maintains patterning of the apical cytoskeleton in sensory hair cells.

Sound transduction occurs in the hair bundle, the apical compartment of sensory hair cells in the inner ear. The hair bundle is formed of actin-based stereocilia aligned in rows of graded heights. It was previously shown that the GNAI-GPSM2 complex is part of a developmental blueprint that defines the polarized organization of the apical cytoskeleton in hair cells, including stereocilia distribution and elongation. Here, we report a role for multiple PDZ domain (MPDZ) protein during apical hair cell morphogenesis in mouse. We show that MPDZ is enriched at the hair cell apical membrane along with MAGUK p55 subfamily member 5 (MPP5/PALS1) and the Crumbs protein CRB3. MPDZ is required there to maintain the proper segregation of apical blueprint proteins, including GNAI-GPSM2. Loss of the blueprint coincides with misaligned stereocilia placement in Mpdz mutant hair cells, and results in permanently misshapen hair bundles. Graded molecular and structural defects along the cochlea can explain the profile of hearing loss in Mpdz mutants, where deficits are most severe at high frequencies.

Hair Cell Protection from Ototoxic Drugs.

Hearing loss is often caused by death of sensory hair cells (HCs) in the inner ear. HCs are vulnerable to some ototoxic drugs, such as aminoglycosides(AGs) and the cisplatin.The most predominant form of drug-induced cell death is apoptosis. Many efforts have been made to protect HCs from cell death after ototoxic drug exposure. These mechanisms and potential targets of HCs protection will be discussed in this review.And we also propose further investigation in the field of HCs necrosis and regeneration, as well as future clinical utilization.

The crosstalk between the Notch, Wnt, and SHH signaling pathways in regulating the proliferation and regeneration of sensory progenitor cells in the mouse cochlea

Sensory hair cells (HCs) are highly susceptible to damage by noise, ototoxic drugs, and aging. Although HCs cannot be spontaneously regenerated in adult mammals, previous studies have shown that signaling pathways are involved in HC regeneration in the damaged mouse cochlea. Here, we used a Notch antagonist (DAPT), a Wnt agonist (QS11), and recombinant Sonic hedgehog (SHH) protein to investigate their concerted actions underlying HC regeneration in the mouse cochlea after neomycin-induced damage both in vivo and in vitro. With DAPT, the numbers of HCs increased, and supporting cell (SC) proliferation was seen in both the intact and damaged cochlear sensory epithelia, while these numbers were unchanged in the presence of QS11. When simultaneously treated with DAPT and QS11, the number of HCs increased dramatically, and much greater SC proliferation was seen in the cochlear epithelium. In transgenic mice with both Notch1 conditional knockout and β-catenin over-expression, cochlear SC proliferation and HC regeneration were more obvious than in either Notch1 knockout or β-catenin over-expressing mice separately. When cochleae were treated with DAPT, QS11, and SHH together, SC proliferation was even greater, and this proliferation was seen in both the HC region and the greater epithelial ridge. High-throughput RNA sequencing was used to identify the differentially expressed genes between all groups, and the results showed that the SHH and Wnt signaling pathways are involved in SC proliferation. Our study suggests that co-regulation of the Notch, Wnt, and SHH signaling pathways promotes extensive cell proliferation and regeneration in the mouse cochlea.

Exosomes as drug delivery vehicles and biomarkers for neurological and auditory systems.

Exosomes are small extracellular membrane particles that play a crucial role in intracellular signaling. Research shows that exosomes have the potential to be used as biomarkers or drug delivery systems in specific organs, such as the neurological system and the inner ear. Exosomes in neurological and auditory systems release different molecules when under stress versus in healthy states, highlighting their potential use as biomarkers in the identification of diseased states. Studies have suggested that exosomes can be harnessed for drug delivery to hard-to-reach organs, such as cochlear sensory hair cells and the brain due to their ability to cross the blood-labyrinth and blood-brain barriers. In this article, we describe the biogenesis, classification, and characterization methods of exosomes. We then discuss recent studies that indicate their potential usage as biomarkers and drug delivery systems to help treat inner ear and neurological disorders.

IGF-1 Haploinsufficiency Causes Age-Related Chronic Cochlear Inflammation and Increases Noise-Induced Hearing Loss

Insulin-like growth factor 1 (IGF-1) deficiency is an ultrarare syndromic human sensorineural deafness. Accordingly, IGF-1 is essential for the postnatal maturation of the cochlea and the correct wiring of hearing in mice. Less severe decreases in human IGF-1 levels have been associated with other hearing loss rare genetic syndromes, as well as with age-related hearing loss (ARHL). However, the underlying mechanisms linking IGF-1 haploinsufficiency with auditory pathology and ARHL have not been studied. Igf1-heterozygous mice express less Igf1 transcription and have 40% lower IGF-1 serum levels than wild-type mice. Along with ageing, IGF-1 levels decreased concomitantly with the increased expression of inflammatory cytokines, Tgfb1 and Il1b, but there was no associated hearing loss. However, noise exposure of these mice caused increased injury to sensory hair cells and irreversible hearing loss. Concomitantly, there was a significant alteration in the expression ratio of pro- and anti-inflammatory cytokines in Igf1+/− mice. Unbalanced inflammation led to the activation of the stress kinase JNK and the failure to activate AKT. Our data show that IGF-1 haploinsufficiency causes a chronic subclinical proinflammatory age-associated state and, consequently, greater susceptibility to stressors. This work provides the molecular bases to further understand hearing disorders linked to IGF-1 deficiency.

Temporal Bone Pathology Secondary to Head Trauma-A Human Temporal Bone Study.

We hypothesize that following head trauma there is a difference in temporal bone (TB) pathology in cases with and without skull fracture. Although conductive, sensorineural, mixed hearing loss, and TB pathology following head trauma have been reported, to our knowledge, there are no studies that have compared the pathology of the TB in cases with and without skull fracture. We analyzed 34 TBs from donors who had a history of head trauma (20 with skull fracture and 14 without fracture), and 25 age-matched controls without clinical or histological evidence of otologic disorders. We documented the presence and location of TB fracture, ossicular injury, and cochlear hemorrhage and evaluated the loss of spiral ganglion cells and sensory hair cells, damage to the stria vascularis, and the presence of endolymphatic hydrops. We found a significant loss of outer hair cells in the upper basal, lower, and upper middle turns of the cochlea (p = 0.009, =0.019, =0.040, respectively), a significant loss of spiral ganglion cells (p = 0.023), and cochlear hemorrhage predominantly in the basal turns secondary to head trauma. Interestingly, these findings were significantly observed in TBs from donors with a history of head trauma without skull fracture. The greatest damage was to the cochlear basal turn. Our findings suggest that head trauma may result in tonotopic high frequency sensorineural hearing loss. TBs from donors with skull fracture have less pathologic changes than those without.

Key Signaling Pathways Regulate the Development and Survival of Auditory Hair Cells.

The loss of auditory sensory hair cells (HCs) is the most common cause of sensorineural hearing loss (SNHL). As the main sound transmission structure in the cochlea, it is necessary to maintain the normal shape and survival of HCs. In this review, we described and summarized the signaling pathways that regulate the development and survival of auditory HCs in SNHL. The role of the mitogen-activated protein kinase (MAPK), phosphoinositide-3 kinase/protein kinase B (PI3K/Akt), Notch/Wnt/Atoh1, calcium channels, and oxidative stress/reactive oxygen species (ROS) signaling pathways are the most relevant. The molecular interactions of these signaling pathways play an important role in the survival of HCs, which may provide a theoretical basis and possible therapeutic interventions for the treatment of hearing loss.

Cochlear apical morphology in toothed whales: Using the pairing hair cell-Deiters' cell as a marker to detect lesions.

The apex or apical region of the cochlear spiral within the inner ear encodes for low-frequency sounds. The disposition of sensory hair cells on the organ of Corti is largely variable in the apical region of mammals, and it does not necessarily follow the typical three-row pattern of outer hair cells (OHCs). As most underwater noise sources contain low-frequency components, we expect to find most lesions in the apical region of the cochlea of toothed whales, in cases of permanent noise-induced hearing loss. To further understand how man-made noise might affect cetacean hearing, there is a need to describe normal morphological features of the apex and document interspecific anatomic variations in cetaceans. However, distinguishing between apical normal variability and hair cell death is challenging. We describe anatomical features of the organ of Corti of the apex in 23 ears from five species of toothed whales (harbor porpoise Phocoena phocoena, spinner dolphin Stenella longirostris, pantropical spotted dolphin Stenella attenuata, pygmy sperm whale Kogia breviceps, and beluga whale Delphinapterus leucas) by scanning electron microscopy and immunofluorescence. Our results showed an initial region where the lowest frequencies are encoded with two or three rows of OHCs, followed by the typical configuration of three OHC rows and three rows of supporting Deiters' cells. Whenever two rows of OHCs were detected, there were usually only two corresponding rows of supporting Deiters' cells, suggesting that the number of rows of Deiters' cells is a good indicator to distinguish between normal and pathological features.

Small-molecule inhibition of Lats kinases may promote Yap-dependent proliferation in postmitotic mammalian tissues

Hippo signaling is an evolutionarily conserved pathway that restricts growth and regeneration predominantly by suppressing the activity of the transcriptional coactivator Yap. Using a high-throughput phenotypic screen, we identified a potent and non-toxic activator of Yap. In vitro kinase assays show that the compound acts as an ATP-competitive inhibitor of Lats kinases—the core enzymes in Hippo signaling. The substance prevents Yap phosphorylation and induces proliferation of supporting cells in the murine inner ear, murine cardiomyocytes, and human Müller glia in retinal organoids. RNA sequencing indicates that the inhibitor reversibly activates the expression of transcriptional Yap targets: upon withdrawal, a subset of supporting-cell progeny exits the cell cycle and upregulates genes characteristic of sensory hair cells. Our results suggest that the pharmacological inhibition of Lats kinases may promote initial stages of the proliferative regeneration of hair cells, a process thought to be permanently suppressed in the adult mammalian inner ear.

How Transmembrane Inner Ear (TMIE) plays role in the auditory system: A mystery to us.

Different cellular mechanisms contribute to the hearing sense, so it is obvious that any disruption in such processes leads to hearing impairment that greatly influences the global economy and quality of life of the patients and their relatives. In the past two decades, transmembrane inner ear (TMIE) protein has received a great deal of research interest because its impairments cause hereditary deafness in humans. This evolutionarily conserved membrane protein contributes to a fundamental complex that plays role in the maintenance and function of the sensory hair cells. Although the critical roles of the TMIE in mechanoelectrical transduction or hearing procedures have been discussed, there are little to no review papers summarizing the roles of the TMIE in the auditory system. In order to fill this gap, herein, we discuss the important roles of this protein in the auditory system including its role in mechanotransduction, olivocochlear synapse, morphology and different signalling pathways; we also review the genotype‐phenotype correlation that can per se show the possible roles of this protein in the auditory system.

Truncation of the otoferlin transmembrane domain alters the development of hair cells and reduces membrane docking.

Release of neurotransmitter from sensory hair cells is regulated by otoferlin. Despite the importance of otoferlin in the auditory and vestibular pathways, the functional contributions of the domains of the protein have not been fully characterized. Using a zebrafish model, we investigated a mutant otoferlin with a stop codon at the start of the transmembrane domain. We found that both the phenotype severity and the expression level of mutant otoferlin changed with the age of the zebrafish. At the early developmental time point of 72 h post fertilization, low expression of the otoferlin mutant coincided with synaptic ribbon deficiencies, reduced endocytosis, and abnormal transcription of several hair cell genes. As development proceeded, expression of the mutant otoferlin increased, and both synaptic ribbons and hair cell transcript levels resembled wild type. However, hair cell endocytosis deficits and abnormalities in the expression of GABA receptors persisted even after up-regulation of mutant otoferlin. Analysis of membrane-reconstituted otoferlin measurements suggests a function for the transmembrane domain in liposome docking. We conclude that deletion of the transmembrane domain reduces membrane docking, attenuates endocytosis, and results in developmental delay of the hair cell.

Intratympanic Lipopolysaccharide Elevates Systemic Fluorescent Gentamicin Uptake in the Cochlea.

Objectives/HypothesisLipopolysaccharide (LPS), a key component of bacterial endotoxins, activates macrophages and triggers the release of inflammatory cytokines in mammalian tissues. Recent studies have shown that intratympanic injection of LPS simulates acute otitis media (AOM) and results in morphological and functional changes in the inner ear. Here we established an AOM mouse model with LPS to investigate the uptake of ototoxic gentamicin in the inner ear, and elucidated the underlying mechanism by focusing on cochlear inflammation as a result of AOM.Study DesignPreclinical rodent animal model.MethodsFluorescently tagged gentamicin (GTTR) was systemically administered to mice with AOM. Iba1‐positive macrophage morphology and inner ear cytokine profile were evaluated by immunofluorescence technique and a mouse cytokine array kit, respectively.ResultsWe observed characteristic symptoms of AOM in the LPS‐treated ears with elevated hearing thresholds indicating a conductive hearing loss. More importantly, the LPS‐induced AOM activated cochlear inflammatory responses, manifested by macrophage infiltration, particularly in the organ of Corti and the spiral ligament, in addition to the up‐regulation of proinflammatory cytokines. Meanwhile, GTTR uptake in the stria vascularis and sensory hair cells from all the LPS‐treated ears was significantly enhanced at 24, 48, and 72‐hour post‐treatment, as the most prominent enhancement was observed in the 48‐hour group.ConclusionIn summary, this study suggests that the pathological cochlea is more susceptible to ototoxic drugs, including aminoglycosides, and justified the clinical concern of aminoglycoside ototoxicity in the AOM treatment. Laryngoscope, 131:E2573–E2582, 2021

Neurog1, Neurod1, and Atoh1 are essential for spiral ganglia, cochlear nuclei, and cochlear hair cell development.

We review the molecular basis of three related basic helix–loop–helix (bHLH) genes (Neurog1, Neurod1, and Atoh1) and upstream regulators Eya1/Six1, Sox2, Pax2, Gata3, Fgfr2b, Foxg1, and Lmx1a/b during the development of spiral ganglia, cochlear nuclei, and cochlear hair cells. Neuronal development requires early expression of Neurog1, followed by its downstream target Neurod1, which downregulates Atoh1 expression. In contrast, hair cells and cochlear nuclei critically depend on Atoh1 and require Neurod1 and Neurog1 expression for various aspects of development. Several experiments show a partial uncoupling of Atoh1/Neurod1 (spiral ganglia and cochlea) and Atoh1/Neurog1/Neurod1 (cochlear nuclei). In this review, we integrate the cellular and molecular mechanisms that regulate the development of auditory system and provide novel insights into the restoration of hearing loss, beyond the limited generation of lost sensory neurons and hair cells.

Intraflagellar transport

Cells need to be able to sense different types of signals, such as chemical and mechanical stimuli, from the extracellular environment in order to properly function. Most eukaryotic cells sense these signals in part through a specialized hair-like organelle, the cilium, that extends from the cell body as a sort of antenna. The signaling and sensory functions of cilia are fundamental during the early stages of embryonic development, when cilia coordinate the establishment of the internal left-right asymmetry that is typical of the vertebrate body. Later, cilia continue to be required for the correct development and function of specific tissues and organs, such as the brain, heart, kidney, liver, and pancreas. Sensory cilia allow us to sense the environment that surrounds us; for instance, we see as a result of the connecting cilia of photoreceptors in our retina, we smell through the sensory cilia at the tips of our olfactory neurons, and we hear thanks to the kinocilia of our sensory hair cells. Motile cilia, which themselves have sensory functions, also work as propeller-like extensions that allow us to breathe because they keep our lungs clean, to reproduce because they propel sperm cells, and even to properly reason because they contribute to the flow of cerebrospinal fluid in our brain ventricles. Not surprisingly, defects in the assembly and function of these tiny organelles result in devastating pathologies, collectively known as ciliopathies (Box 1). Thus, the proper function of cilia is fundamental for human health.

Adaptive cell invasion maintains lateral line organ homeostasis in response to environmental changes.

Mammalian inner ear and fish lateral line sensory hair cells (HCs) detect fluid motion to transduce environmental signals. Actively maintained ionic homeostasis of the mammalian inner ear endolymph is essential for HC function. In contrast, fish lateral line HCs are exposed to the fluctuating ionic composition of the aqueous environment. Using lineage labeling, invivo time-lapse imaging and scRNA-seq, we discovered highly motile skin-derived cells that invade mature mechanosensory organs of the zebrafish lateral line and differentiate into Neuromast-associated (Nm) ionocytes. This invasion is adaptive as it is triggered by environmental fluctuations. Our discovery of Nm ionocytes challenges the notion of an entirely placodally derived lateral line and identifies Nm ionocytes as likely regulators of HC function possibly by modulating the ionic microenvironment. Nm ionocytes provide an experimentally accessible invivo system to study cell invasion and migration, as well as the physiological adaptation of vertebrate organs to changing environmental conditions.

Non-Canonical Wnt Signaling Regulates Cochlear Outgrowth and Planar Cell Polarity via Gsk3β Inhibition.

During development, sensory hair cells (HCs) in the cochlea assemble a stereociliary hair bundle on their apical surface with planar polarized structure and orientation. We have recently identified a non-canonical, Wnt/G-protein/PI3K signaling pathway that promotes cochlear outgrowth and coordinates planar polarization of the HC apical cytoskeleton and alignment of HC orientation across the cochlear epithelium. Here, we determined the involvement of the kinase Gsk3β and the small GTPase Rac1 in non-canonical Wnt signaling and its regulation of the planar cell polarity (PCP) pathway in the cochlea. We provided the first in vivo evidence for Wnt regulation of Gsk3β activity via inhibitory Ser9 phosphorylation. Furthermore, we carried out genetic rescue experiments of cochlear defects caused by blocking Wnt secretion. We showed that cochlear outgrowth was partially rescued by genetic ablation of Gsk3β but not by expression of stabilized β-catenin; while PCP defects, including hair bundle polarity and junctional localization of the core PCP proteins Fzd6 and Dvl2, were partially rescued by either Gsk3β ablation or constitutive activation of Rac1. Our results identify Gsk3β and likely Rac1 as downstream components of non-canonical Wnt signaling and mediators of cochlear outgrowth, HC planar polarity, and localization of a subset of core PCP proteins in the cochlea.

Regulation of otocyst patterning by Tbx2 and Tbx3 is required for inner ear morphogenesis in the mouse.

All epithelial components of the inner ear, including sensory hair cells and innervating afferent neurons, arise by patterning and differentiation of epithelial progenitors residing in a simple sphere, the otocyst. Here, we identify the transcriptional repressors TBX2 and TBX3 as novel regulators of these processes in the mouse. Ablation of Tbx2 from the otocyst led to cochlear hypoplasia, whereas loss of Tbx3 was associated with vestibular malformations. The loss of function of both genes (Tbx2/3cDKO) prevented inner ear morphogenesis at midgestation, resulting in indiscernible cochlear and vestibular structures at birth. Morphogenetic impairment occurred concomitantly with increased apoptosis in ventral and lateral regions of Tbx2/3cDKO otocysts around E10.5. Expression analyses revealed partly disturbed regionalisation, and a posterior-ventral expansion of the neurogenic domain in Tbx2/3cDKO otocysts at this stage. We provide evidence that repression of FGF signalling by TBX2 is important to restrict neurogenesis to the anterior-ventral otocyst and implicate another T-box factor, TBX1, as a crucial mediator in this regulatory network.

Identification of a series of hair-cell MET channel blockers that protect against aminoglycoside-induced ototoxicity.

To identify small molecules that shield mammalian sensory hair cells from the ototoxic side effects of aminoglycoside antibiotics, 10,240 compounds were initially screened in zebrafish larvae, selecting for those that protected lateral-line hair cells against neomycin and gentamicin. When the 64 hits from this screen were retested in mouse cochlear cultures, 8 protected outer hair cells (OHCs) from gentamicin in vitro without causing hair-bundle damage. These 8 hits shared structural features and blocked, to varying degrees, the OHC’s mechano-electrical transducer (MET) channel, a route of aminoglycoside entry into hair cells. Further characterization of one of the strongest MET channel blockers, UoS-7692, revealed it additionally protected against kanamycin and tobramycin and did not abrogate the bactericidal activity of gentamicin. UoS-7692 behaved, like the aminoglycosides, as a permeant blocker of the MET channel; significantly reduced gentamicin–Texas red loading into OHCs; and preserved lateral-line function in neomycin-treated zebrafish. Transtympanic injection of UoS-7692 protected mouse OHCs from furosemide/kanamycin exposure in vivo and partially preserved hearing. The results confirmed the hair-cell MET channel as a viable target for the identification of compounds that protect the cochlea from aminoglycosides and provide a series of hit compounds that will inform the design of future otoprotectants.

Transcription Factor Reprogramming in the Inner Ear: Turning on Cell Fate Switches to Regenerate Sensory Hair Cells.

Non-mammalian vertebrates can restore their auditory and vestibular hair cells naturally by triggering the regeneration of adjacent supporting cells. The transcription factor ATOH1 is a key regulator of hair cell development and regeneration in the inner ear. Following the death of hair cells, supporting cells upregulate ATOH1 and give rise to new hair cells. However, in the mature mammalian cochlea, such natural regeneration of hair cells is largely absent. Transcription factor reprogramming has been used in many tissues to convert one cell type into another, with the long-term hope of achieving tissue regeneration. Reprogramming transcription factors work by altering the transcriptomic and epigenetic landscapes in a target cell, resulting in a fate change to the desired cell type. Several studies have shown that ATOH1 is capable of reprogramming cochlear non-sensory tissue into cells resembling hair cells in young animals. However, the reprogramming ability of ATOH1 is lost with age, implying that the potency of individual hair cell-specific transcription factors may be reduced or lost over time by mechanisms that are still not clear. To circumvent this, combinations of key hair cell transcription factors have been used to promote hair cell regeneration in older animals. In this review, we summarize recent findings that have identified and studied these reprogramming factor combinations for hair cell regeneration. Finally, we discuss the important questions that emerge from these findings, particularly the feasibility of therapeutic strategies using reprogramming factors to restore human hearing in the future.