Mechanosensory Hair Cells Research Articles

Celastrol inhibits aminoglycoside-induced ototoxicity via heat shock protein 32

Hearing loss is often caused by death of the mechanosensory hair cells of the inner ear. Hair cells are susceptible to death caused by aging, noise trauma, and ototoxic drugs, including the aminoglycoside antibiotics and the antineoplastic agent cisplatin. Ototoxic drugs result in permanent hearing loss for over 500 000 Americans annually. We showed previously that induction of heat shock proteins (HSPs) inhibits both aminoglycoside- and cisplatin-induced hair cell death in whole-organ cultures of utricles from adult mice. In order to begin to translate these findings into a clinical therapy aimed at inhibiting ototoxic drug-induced hearing loss, we have now examined a pharmacological HSP inducer, celastrol. Celastrol induced upregulation of HSPs in utricles, and it provided significant protection against aminoglycoside-induced hair cell death in vitro and in vivo. Moreover, celastrol inhibited hearing loss in mice receiving systemic aminoglycoside treatment. Our data indicate that the major heat shock transcription factor HSF-1 is not required for celastrol-mediated protection. HSP32 (also called heme oxygenase-1, HO-1) is the primary mediator of the protective effect of celastrol. HSP32/HO-1 inhibits pro-apoptotic c-Jun N-terminal kinase (JNK) activation and hair cell death. Taken together, our data indicate that celastrol inhibits aminoglycoside ototoxicity via HSP32/HO-1 induction.

Fucoidan promotes mechanosensory hair cell regeneration following amino glycoside-induced cell death

Objective Lateral line system of the zebrafish is a useful model for study of hair cell toxicity and regeneration. We found that low molecular weight fucoidan (LMWF) stimulated the regeneration of mechanosensory hair cells after neomycin-induced cell death in zebrafish lateral line. The aims of this study were to quantify the regenerative effects of LMWF and determine their relationship to the Notch and FGF signaling pathways. Methods Wild-type zebrafish and three different transgenic zebrafish lines ( Pou4f3::GFP, scm1::GFP, and ET20::GFP) were used. At 4.5–6 days post-fertilization, lateral line hair cells of larvae were eliminated using neomycin (500 μM). Larvae were then treated with LMWF. Neuromasts were observed using confocal microscopy. Stereocilia morphology was observed using scanning electron microscopy, and the location and status of regeneration was assessed using 5-bromo-2-deoxyuridine (BrdU) incorporation. Results Hair cells damaged by neomycin treatment regenerated faster in wild-type and Pou4f3::GFP larvae treated with LMWF (50 μg/ml) than in untreated controls. LMWF also enhanced the regeneration of supporting cells in scm1::GFP and ET20::GFP larvae. Increased numbers of BrdU-labeled cells were found after LMWF treatment in neuromast regions corresponding to internal and peripheral supporting cells. The effect of LMWF was mimicked by the Notch signaling inhibitor N-[N-(3,5-difluorophenacetyl)-1-alanyl]-S-phenylglycine t-butyl ester (DAPT), but the effects of LMWF and DAPT were not additive. Conclusion LMWF enhances the regeneration of hair cells damaged by neomycin. The mechanism may involve the Notch signaling pathway. LMWF shows promise as a therapeutic agent for hearing and balance disorders.

Balancing cell numbers during organogenesis: Six1a differentially affects neurons and sensory hair cells in the inner ear

While genes involved in the differentiation of the mechanosensory hair cells and the neurons innervating them have been identified, genes involved in balancing their relative numbers remain unknown. Six1a plays a dual role by promoting hair cell fate while inhibiting neuronal fate in these two lineages. Genes homologous to six1a act as either transcriptional activators or repressors, depending on the partners with which they interact. By assaying the in vivo and in vitro effects of mutations in presumptive protein-protein interacting and DNA-binding domains of Six1a, we show that, in the developing zebrafish inner ear, Six1a promotes hair cell fate by acting as a transcriptional activator and inhibits neuronal fate by acting as a transcriptional repressor. We also identify several potential partners for Six1a that differ between these two lineages. The dual role of Six1a in the developing otocyst provides a mechanism for balancing the relative number of hair cells and neurons during organogenesis of the inner ear.

The paratympanic organ: a barometer and altimeter in the middle ear of birds?

A century has passed since the discovery of the paratympanic organ (PTO), a mechanoreceptive sense organ in the middle ear of birds and other tetrapods. This luminal organ contains a sensory epithelium with typical mechanosensory hair cells and may function as a barometer and altimeter. The organ is arguably the most neglected sense organ in living tetrapods. The PTO is believed to be homologous to a lateral line sense organ, the spiracular sense organ of nonteleostean fishes. Our review summarizes the current state of knowledge of the PTO and draws attention to the astounding lack of information about the unique and largely unexplored sensory modality of barometric perception.

Besides Huntington's disease, does brain-type creatine kinase play a role in other forms of hearing impairment resulting from a common pathological cause?

Hearing impairment following cochlear damage due to noise trauma, ototoxicity caused by aminoglycoside antibiotics, or age-related cochlear degeneration was linked to a common pathogenesis involving the formation of reactive oxygen species (ROS). Cochleae are more vulnerable to oxidative stress than other organs because of the high metabolic demands of their mechanosensory hair cells in response to sound stimulation. We recently showed that patients and mice with Huntington's disease (HD) have hearing impairment and that the dysregulated phosphocreatine (PCr)-creatine kinase (CK) system may account for this auditory dysfunction. Given the importance of noninvasive biomarkers and the easy access of hearing tests, the symptom of hearing loss in HD patients may serve as a useful clinical indicator of disease onset and progression of HD. We also showed that dietary creatine supplementation rescued the impaired PCr-CK system and improved the expression of cochlear brain-type creatine kinase (CKB) in HD mice, thereby restoring their hearing. Because creatine is an antioxidant, we postulated that creatine might enhance expression of CKB by reducing oxidative stress. In addition to HD-related hearing impairment, inferior CKB expression and/or an impaired PCr-CK system may also play an important role in other hearing impairments caused by elevated levels of ROS. Most importantly, dietary supplements may be beneficial to patients with these hearing deficiencies.

In vitro cultured primary cells from a human utricle explant possesses hair cell like characteristics

The utricle is the enlarged portion of the membranous labyrinth of the inner ear and is essential for balance. It comprises of fine hair cells (mechanoreceptors), supporting cells and calcareous otoliths. Utricle cells are considered to be post-mitotic and possess a limited capacity for regeneration. Unlike birds and reptiles, mammalian mechanosensory hair cells do not regenerate. The in vitro culture of primary cells from the utricle and other inner ear structures of mammals have proven difficult. Presented here for the first time is the culture of primary cells derived from an explant of an adult human utricle, without any intervention or manipulation. Cells were proliferative until cellular quiescence occurred during passage six. Cell morphology was atypical of epithelial cells, appearing as a homogenous, slightly elongated population. Analysis of cultured utricle cells by immunofluorescent staining (IF) and reverse transcriptase polymerase chain reaction (RT-PCR) have shown these cells to possess epithelial (Epithelium-specific ets-1 (ESE-1)), supporting hair cell (p27(Kip1)), and hair cell specific (Atoh1 and Myosin VI) markers. Additionally, RT-PCR revealed positive gene expression for the proliferation control marker fibroblast growth factor receptor 1 (FGFR1) and negative gene expression for E-cadherin (CDH1), a vestibular cell differentiation marker.

Regulation of PCDH15 function in mechanosensory hair cells by alternative splicing of the cytoplasmic domain

Protocadherin 15 (PCDH15) is expressed in hair cells of the inner ear and in photoreceptors of the retina. Mutations in PCDH15 cause Usher Syndrome (deaf-blindness) and recessive deafness. In developing hair cells, PCDH15 localizes to extracellular linkages that connect the stereocilia and kinocilium into a bundle and regulate its morphogenesis. In mature hair cells, PCDH15 is a component of tip links, which gate mechanotransduction channels. PCDH15 is expressed in several isoforms differing in their cytoplasmic domains, suggesting that alternative splicing regulates PCDH15 function in hair cells. To test this model, we generated three mouse lines, each of which lacks one out of three prominent PCDH15 isoforms (CD1, CD2 and CD3). Surprisingly, mice lacking PCDH15-CD1 and PCDH15-CD3 form normal hair bundles and tip links and maintain hearing function. Tip links are also present in mice lacking PCDH15-CD2. However, PCDH15-CD2-deficient mice are deaf, lack kinociliary links and have abnormally polarized hair bundles. Planar cell polarity (PCP) proteins are distributed normally in the sensory epithelia of the mutants, suggesting that PCDH15-CD2 acts downstream of PCP components to control polarity. Despite the absence of kinociliary links, vestibular function is surprisingly intact in the PCDH15-CD2 mutants. Our findings reveal an essential role for PCDH15-CD2 in the formation of kinociliary links and hair bundle polarization, and show that several PCDH15 isoforms can function redundantly at tip links.

Imaging electrical resonance in hair cells

The mechanosensory hair cells of many auditory receptor organs are tuned by an electrical resonance that increases their responses to stimulation over a narrow band of frequencies. The small oscillations of membrane potential characteristic of this phenomenon have previously been detectable only through intracellular electrode measurements, which are laborious and preclude analysis at the level of an entire sensory organ. We used a voltage-sensitive dye to image hair-cell electrical resonance in an intact preparation of the bullfrog's sacculus, a receptor organ sensitive to low-frequency seismic and auditory stimuli. Imaging revealed distinct populations of hair cells whose resonant response varied with the frequency of transepithelial electrical stimulation. Most of the hair cells in the saccular epithelium in vitro were electrically tuned to stimulation at 25-50 Hz. The frequency dependence of the fluorescence signal was sensitive to pharmacological blockade of large-conductance Ca(2+)-sensitive K(+) channels and to enzymatic digestion. At an elevated concentration of Ca(2+), we observed transient fluorescence signals that probably represented action potentials. The stroboscopic imaging and analysis techniques described here present a general approach for studying subthreshold oscillations in electrically excitable cells.

Identification of genes concordantly expressed withAtoh1during inner ear development

The inner ear is composed of a cochlear duct and five vestibular organs in which mechanosensory hair cells play critical roles in receiving and relaying sound and balance signals to the brain. To identify novel genes associated with hair cell differentiation or function, we analyzed an archived gene expression dataset from embryonic mouse inner ear tissues. Since atonal homolog 1a (Atoh1) is a well known factor required for hair cell differentiation, we searched for genes expressed in a similar pattern with Atoh1 during inner ear development. The list from our analysis includes many genes previously reported to be involved in hair cell differentiation such as Myo6, Tecta, Myo7a, Cdh23, Atp6v1b1, and Gfi1. In addition, we identified many other genes that have not been associated with hair cell differentiation, including Tekt2, Spag6, Smpx, Lmod1, Myh7b, Kif9, Ttyh1, Scn11a and Cnga2. We examined expression patterns of some of the newly identified genes using real-time polymerase chain reaction and in situ hybridization. For example, Smpx and Tekt2, which are regulators for cytoskeletal dynamics, were shown specifically expressed in the hair cells, suggesting a possible role in hair cell differentiation or function. Here, by reanalyzing archived genetic profiling data, we identified a list of novel genes possibly involved in hair cell differentiation.

Molecular dissection of the migrating posterior lateral line primordium during early development in zebrafish

BackgroundDevelopment of the posterior lateral line (PLL) system in zebrafish involves cell migration, proliferation and differentiation of mechanosensory cells. The PLL forms when cranial placodal cells delaminate and become a coherent, migratory primordium that traverses the length of the fish to form this sensory system. As it migrates, the primordium deposits groups of cells called neuromasts, the specialized organs that contain the mechanosensory hair cells. Therefore the primordium provides both a model for studying collective directional cell migration and the differentiation of sensory cells from multipotent progenitor cells.ResultsThrough the combined use of transgenic fish, Fluorescence Activated Cell Sorting and microarray analysis we identified a repertoire of key genes expressed in the migrating primordium and in differentiated neuromasts. We validated the specific expression in the primordium of a subset of the identified sequences by quantitative RT-PCR, and by in situ hybridization. We also show that interfering with the function of two genes, f11r and cd9b, defects in primordium migration are induced. Finally, pathway construction revealed functional relationships among the genes enriched in the migrating cell population.ConclusionsOur results demonstrate that this is a robust approach to globally analyze tissue-specific expression and we predict that many of the genes identified in this study will show critical functions in developmental events involving collective cell migration and possibly in pathological situations such as tumor metastasis.

Atp2b1a regulates Ca2+ export during differentiation and regeneration of mechanosensory hair cells in zebrafish

The molecular mechanisms of development of mechanosensory hair cells have been tackled successfully due to in vivo studies in the zebrafish lateral line. The enhancer trap (ET) transgenic line, SqET4 was instrumental in these studies even despite a lack of a link of its GFP expression pattern to a particular gene(s). We mapped the Tol2 transposon insertion of the SqET4 transgenics onto Chr. 4 next to a gene encoding Atp2b1a (Pmca1) – one of the four PMCAs acting to export Ca2+ from a cell. atp2b1a expression recapitulates that of GFP during the development of mechanoreceptors of the inner ear and lateral line. atp2b1a expression correlates with the regeneration of these cells. Thus, SqET4 represents the Tg:atp2b1a-GFP line, which links Ca2+ metabolism and the differentiation of mechanoreceptors. The morpholino-mediated knockdown of atp2b1a blocks Ca2+ export and affects the division of hair cell progenitors, resulting in their accumulation. Under the control of a master gene of hair cells, Atoh1a, Atp2b1a functions during progenitor cell proliferation and hair cell differentiation. Given the similarity between the phenotypes of atp2b1a morphants and embryos treated with the pan-PMCA inhibitor 5(6)-carboxyeosin, Atp2b1a emerges as member of the Atp2b family responsible for Ca2+ export during the development of hair cells.

The interplay between active hair bundle motility and electromotility in the cochlea

The cochlear amplifier is a nonlinear active process providing the mammalian ear with its extraordinary sensitivity, large dynamic range and sharp frequency tuning. While there is much evidence that amplification results from active force generation by mechanosensory hair cells, there is debate about the cellular processes behind nonlinear amplification. Outer hair cell electromotility has been suggested to underlie the cochlear amplifier. However, it has been shown in frog and turtle that spontaneous movements of hair bundles endow them with a nonlinear response with increased sensitivity that could be the basis of amplification. The present work shows that the properties of the cochlear amplifier could be understood as resulting from the combination of both hair bundle motility and electromotility in an integrated system that couples these processes through the geometric arrangement of hair cells embedded in the cochlear partition. In this scenario, the cochlear partition can become a dynamic oscillator which in the vicinity of a Hopf bifurcation exhibits all the key properties of the cochlear amplifier. The oscillatory behavior and the nonlinearity are provided by active hair bundles. Electromotility is largely linear but produces an additional feedback that allows hair bundle movements to couple to basilar membrane vibrations.

The cell biology of hearing

Mammals have an astonishing ability to sense and discriminate sounds of different frequencies and intensities. Fundamental for this process are mechanosensory hair cells in the inner ear that convert sound-induced vibrations into electrical signals. The study of genes that are linked to deafness has provided insights into the cell biological mechanisms that control hair cell development and their function as mechanosensors.

Structure of the N terminus of cadherin 23 reveals a new adhesion mechanism for a subset of cadherin superfamily members

The cadherin superfamily encodes more than 100 receptors with diverse functions in tissue development and homeostasis. Classical cadherins mediate adhesion by binding interactions that depend on their N-terminal extracellular cadherin (EC) domains, which swap N-terminal beta-strands. Sequence alignments suggest that the strand-swap binding mode is not commonly used by functionally divergent cadherins. Here, we have determined the structure of the EC1-EC2 domains of cadherin 23 (CDH23), which binds to protocadherin 15 (PCDH15) to form tip links of mechanosensory hair cells. Unlike classical cadherins, the CDH23 N terminus contains polar amino acids that bind Ca(2+). The N terminus of PCDH15 also contains polar amino acids. Mutations in polar amino acids within EC1 of CDH23 and PCDH15 abolish interaction between the two cadherins. PCDH21 and PCDH24 contain similarly charged N termini, suggesting that a subset of cadherins share a common interaction mechanism that differs from the strand-swap binding mode of classical cadherins.

From shared lineage to distinct functions: the development of the inner ear and epibranchial placodes

The inner ear and the epibranchial ganglia constitute much of the sensory system in the caudal vertebrate head. The inner ear consists of mechanosensory hair cells, their neurons, and structures necessary for sound and balance sensation. The epibranchial ganglia are knots of neurons that innervate and relay sensory signals from several visceral organs and the taste buds. Their development was once thought to be independent, in line with their independent functions. However, recent studies indicate that both systems arise from a morphologically distinct common precursor domain: the posterior placodal area. This review summarises recent studies into the induction, morphogenesis and innervation of these systems and discusses lineage restriction and cell specification in the context of their common origin.

Transfection of Mouse Cochlear Explants by Electroporation

The sensory epithelium of the mammalian inner ear, also referred to as the organ of Corti, is a remarkable structure comprised of highly ordered rows of mechanosensory hair cells and non-sensory supporting cells located within the coiled cochlea. This unit describes an in vitro explant culture technique that can be coupled with gene transfer via electroporation to study the effects of altering gene expression during development of the organ of Corti. While the protocol is largely focused on embryonic cochlea, the same basic protocol can be used on cochleae from mice as old as P5.

MicroRNA-183 Family Members Regulate Sensorineural Fates in the Inner Ear

Members of the microRNA (miRNA) 183 family (miR-183, miR-96, and miR-182) are expressed abundantly in specific sensory cell types in the eye, nose, and inner ear. In the inner ear, expression is robust in the mechanosensory hair cells and weak in the associated statoacoustic ganglion (SAG) neurons; both cell types can share a common lineage during development. Recently, dominant-progressive hearing loss in humans and mice was linked to mutations in the seed region of miR-96, with associated defects in both development and maintenance of hair cells in the mutant mice. To understand how the entire triplet functions in the development of mechanosensory hair cells and neurons of the inner ear, we manipulated the levels of these miRNAs in zebrafish embryos using synthesized miRNAs and antisense morpholino oligonucleotides (MOs). Overexpression of miR-96 or miR-182 induces duplicated otocysts, ectopic or expanded sensory patches, and extra hair cells, whereas morphogenesis of the SAG is adversely affected to different degrees. In contrast, knockdown of miR-183, miR-96, and miR-182 causes reduced numbers of hair cells in the inner ear, smaller SAGs, defects in semicircular canals, and abnormal neuromasts on the posterior lateral line. However, the prosensory region of the posterior macula, where the number of hair cells is reduced by approximately 50%, is not significantly impaired. Our findings suggest both distinct and common roles for the three miRNAs in cell-fate determination in the inner ear, and these principles might apply to development of other sensory organs.

Chemical Screening for Hair Cell Loss and Protection in the Zebrafish Lateral Line

In humans, most hearing loss results from death of hair cells, the mechanosensory receptors of the inner ear. Two goals of current hearing research are to protect hair cells from degeneration and to regenerate new hair cells, replacing those that are lost due to aging, disease, or environmental challenges. One limitation of research in the auditory field has been the relative inaccessibility of the mechanosensory systems in the inner ear. Zebrafish possess hair cells in both their inner ear and their lateral line system that are morphologically and functionally similar to human hair cells. The external location of the mechanosensory hair cells in the lateral line and the ease of in vivo labeling and imaging make the zebrafish lateral line a unique system for the study of hair cell toxicity, protection, and regeneration. This review focuses on the lateral line system as a model for understanding loss and protection of mechanosensory hair cells. We discuss chemical screens to identify compounds that induce hair cell loss and others that protect hair cells from known toxins and the potential application of these screens to human medicine.

Drug screening for hearing loss: Using the zebrafish lateral line to screen for drugs that prevent and cause hearing loss

Several animal models have been used for the study of mechanosensory hair cells and hearing loss. Because of the difficulty of tissue acquisition and large animal size, these traditional models are impractical for high-throughput screening. The zebrafish has emerged as a powerful animal model for screening drugs that cause and prevent hair cell death. The unique characteristics of the zebrafish enable rapid in vivo imaging of hair cells and hair cell death. We have used this model to screen for and identify multiple drugs that protect hair cells from aminoglycoside-induced death. The identification of multiple drugs and drug-like compounds that inhibit multiple hair cell death pathways might enable the development of protective cocktails to achieve complete hair cell protection.

Sox2 Induces Neuronal Formation in the Developing Mammalian Cochlea

In the cochlea, spiral ganglion neurons play a critical role in hearing as they form the relay between mechanosensory hair cells in the inner ear and cochlear nuclei in the brainstem. The proneural basic helix-loop-helix transcription factors Neurogenin1 (Neurog1) and NeuroD1 have been shown to be essential for the development of otocyst-derived inner ear sensory neurons. Here, we show neural competence of nonsensory epithelial cells in the cochlea, as ectopic expression of either Neurog1 or NeuroD1 results in the formation of neuronal cells. Since the high-mobility-group type transcription factor Sox2, which is also known to play a role in neurogenesis, is expressed in otocyst-derived neural precursor cells and later in the spiral ganglion neurons along with Neurog1 and NeuroD1, we used both gain- and loss-of-function experiments to examine the role of Sox2 in spiral ganglion neuron formation. We demonstrate that overexpression of Sox2 results in the production of neurons, suggesting that Sox2 is sufficient for the induction of neuronal fate in nonsensory epithelial cells. Furthermore, spiral ganglion neurons are absent in cochleae from Sox2(Lcc/Lcc) mice, indicating that Sox2 is also required for neuronal formation in the cochlea. Our results indicate that Sox2, along with Neurog1 and NeuroD1, are sufficient to induce a neuronal fate in nonsensory regions of the cochlea. Finally, we demonstrate that nonsensory cells within the cochlea retain neural competence through at least the early postnatal period.