Mechanoelectrical Transducer Current Research Articles

AAV-mediated rescue of Eps8 expression in vivo restores hair-cell function in a mouse model of recessive deafness

The transduction of acoustic information by hair cells depends upon mechanosensitive stereociliary bundles that project from their apical surface. Mutations or absence of the stereociliary protein EPS8 cause deafness in humans and mice, respectively. Eps8 knockout mice (Eps8−/−) have hair cells with immature stereocilia and fail to become sensory receptors. Here, we show that exogenous delivery of Eps8 using Anc80L65 in P1–P2 Eps8−/− mice in vivo rescued the hair bundle structure of apical-coil hair cells. Rescued hair bundles correctly localize EPS8, WHIRLIN, MYO15, and BAIAP2L2, and generate normal mechanoelectrical transducer currents. Inner hair cells with normal-looking stereocilia re-expressed adult-like basolateral ion channels (BK and KCNQ4) and have normal exocytosis. The number of hair cells undergoing full recovery was not sufficient to rescue hearing in Eps8−/− mice. Adeno-associated virus (AAV)-transduction of P3 apical-coil and P1–P2 basal-coil hair cells does not rescue hair cells, nor does Anc80L65-Eps8 delivery in adult Eps8−/− mice. We propose that AAV-induced gene-base therapy is an efficient strategy to recover the complex hair-cell defects in Eps8−/− mice. However, this therapeutic approach may need to be performed in utero since, at postnatal ages, Eps8−/− hair cells appear to have matured or accumulated damage beyond the point of repair.

Minimal-invasive enhancement of auditory perception by terahertz wave modulation

Hearing impairment is a common disease affecting a substantial proportion of the global population. Currently, the most effective clinical treatment for patients with sensorineural deafness is to implant an artificial electronic cochlea. However, the improvements to hearing perception are variable and limited among healthy subjects. Moreover, cochlear implants have disadvantages, such as crosstalk derived from the currents that spread into non-target tissue between the electrodes. Here, in this work, we describe terahertz wave modulation, a new and minimally invasive technology that can enhance hearing perception in animals by reversible modulation of currents in cochlear hair cells. Using single-cell electrophysiology, guinea pig audiometry, and molecular dynamics simulations, we show that THM can reversibly increase mechano-electrical transducer currents (∼ 50% higher) and voltage-gated K+ currents in cochlear hair cells through collective resonance of −C=O groups. In addition, measurement of auditory brainstem response (ABR) in guinea pigs treated with THM indicated a ~ 10 dB increase in hearing sensitivity. This study thus reports a new method of highly spatially selective hearing enhancement without introducing any exogeneous gene, which has potential applications for treatment of hearing disorders as well as several other areas of neuroscience.

Preventing autosomal-dominant hearing loss in Bth mice with CRISPR/CasRx-based RNA editing

CRISPR/RfxCas13d (CasRx) editing system can specifically and precisely cleave single-strand RNAs, which is a promising treatment for various disorders by downregulation of related gene expression. Here, we tested this RNA-editing approach on Beethoven (Bth) mice, an animal model for human DFNA36 due to a point mutation in Tmc1. We first screened 30 sgRNAs in cell cultures and found that CasRx with sgRNA3 reduced the Tmc1Bth transcript by 90.8%, and the Tmc1 wild type transcript (Tmc1+) by 44.3%. We then injected a newly developed AAV vector (AAV-PHP.eB) based CasRx into the inner ears of neonatal Bth mice, and we found that Tmc1Bth was reduced by 70.2% in 2 weeks with few off-target effects in the whole transcriptome. Consistently, we found improved hair cell survival, rescued hair bundle degeneration, and reduced mechanoelectrical transduction current. Importantly, the hearing performance, measured in both ABR and DPOAE thresholds, was improved significantly in all ages over 8 weeks. We, therefore, have validated the CRISPR/CasRx-based RNA editing strategy in treating autosomal-dominant hearing loss, paving way for its further application in many other hereditary diseases in hearing and beyond.

Grxcr1 regulates hair bundle morphogenesis and is required for normal mechanoelectrical transduction in mouse cochlear hair cells.

Tasmanian devil (tde) mice are deaf and exhibit circling behaviour. Sensory hair cells of mutants show disorganised hair bundles with abnormally thin stereocilia. The origin of this mutation is the insertion of a transgene which disrupts expression of the Grxcr1 (glutaredoxin cysteine rich 1) gene. We report here that Grxcr1 exons and transcript sequences are not affected by the transgene insertion in tde homozygous (tde/tde) mice. Furthermore, 5’RACE PCR experiments showed the presence of two different transcripts of the Grxcr1 gene, expressed in both tde/tde and in wild-type controls. However, quantitative analysis of Grxcr1 transcripts revealed a significantly decreased mRNA level in tde/tde mice. The key stereociliary proteins ESPN, MYO7A, EPS8 and PTPRQ were distributed in hair bundles of homozygous tde mutants in a similar pattern compared with control mice. We found that the abnormal morphology of the stereociliary bundle was associated with a reduction in the size and Ca2+-sensitivity of the mechanoelectrical transducer (MET) current. We propose that GRXCR1 is key for the normal growth of the stereociliary bundle prior to the onset of hearing, and in its absence hair cells are unable to mature into fully functional sensory receptors.

BAIAP2L2 is required for the maintenance of mechanotransducing stereocilia of cochlear hair cells.

Stereocilia are actin-based cell protrusions of inner ear hair cells that play an essential role in mechano-electrical transduction (MET). Stereocilia are organized into several rows of increasing heights with the MET protein complex localized at the tips of shorter row stereocilia. At the tips of shorter row mechanotransducing stereocilia also resides a so-called "row 2 protein complex" whose dysfunction causes degeneration of the mechanotransducing stereocilia. In the present work, we show that BAIAP2L2 is localized at the tips of shorter row stereocilia in neonatal and adult mouse cochlear hair cells. Baiap2l2 inactivation causes degeneration of the mechanotransducing stereocilia, which eventually leads to profound hearing loss in mice of either sex. Consistently, electrophysiology and FM 1-43FX dye uptake results confirm that MET currents are compromised in Baiap2l2 knockout mice. Moreover, BAIAP2L2 binds to known row 2 complex components EPS8L2, TWF2, and CAPZB2, and the stereociliary tip localization of CAPZB2 is dependent on functional BAIAP2L2. Interestingly, BAIAP2L2 also binds to CIB2, a known MET complex component, and the stereociliary tip localization of BAIAP2L2 is abolished in Cib2 knockout mice. In conclusion, our present data suggest that BAIAP2L2 is a row 2 complex component, and is required for the maintenance of mechanotransducing stereocilia. Meanwhile, specific MET components such as CIB2 might play a direct role in stereocilia maintenance through binding to BAIAP2L2.

Quantifying entropy production in active fluctuations of the hair-cell bundle from time irreversibility and uncertainty relations

We introduce lower bounds for the rate of entropy production of an active stochastic process by quantifying the irreversibility of stochastic traces obtained from mesoscopic degrees of freedom. Our measures of irreversibility reveal signatures of time’s arrow and provide bounds for entropy production even in the case of active fluctuations that have no drift. We apply these irreversibility measures to experimental recordings of spontaneous hair-bundle oscillations in mechanosensory hair cells from the ear of the bullfrog. By analyzing the fluctuations of only the tip position of hair bundles, we reveal irreversibility in active oscillations and estimate an associated rate of entropy production of at least ∼3k B/s, on average. Applying thermodynamic uncertainty relations, we predict that measuring both the tip position of the hair bundle and the mechano-electrical transduction current that enters the hair cell leads to tighter lower bounds for the rate of entropy production, up to ∼103 k B/s in the oscillatory regime.

Disrupting ELKIN1-Dependent Mechanoelectrical Transduction Modulates Cell-Cell Interactions in Organotypic Tumour Spheroids

In the tumour microenvironment, cells within tumour masses can interact with neighbouring cells as well as the surrounding stroma. During melanoma progression, there are many changes to the mechanical properties of the cells and the surrounding tissue, and these mechanical perturbations can drive disease progression. We are studying how cells sense these mechanical changes with a focus on mechanoelectrical transduction: the channel-mediated conversion of mechanical inputs into an electrical signal. Mechanoelectrical transduction currents can be evoked in melanoma cells and in the secondary melanoma cell line, WM266-4, the ablation of TMEM87a/ELKIN1 led to a reduction in mechanically activated (MA) ion channel activity and the modulation of cell migration. To investigate how ELKIN1 impacts cell-cell interactions, we visualised cell patterning and dissociation using organotypic spheroids to mimic a tumour mass. in ELKIN1 deletion mutants, there is a significant increase in cell dissociation from the spheroids, suggesting that ELKIN1 plays a role in modulating cell-cell interactions. in chimeric spheroids formed from WT and ELKIN1-KO cells, we observed that the WT cells sorted to the core of the spheroid, whereas ELKIN1-KO cells sorted to the exterior of the spheroid. These functional changes were accompanied by changes to cell-cell and cell-substrate attachment properties. These data highlight that ELKIN1-dependent mechanoelectrical transduction influences cell-cell and cell-substrate interactions. in contrast, disruption of the expression of PIEZO1 (a well-characterised MA ion channel) did not exhibit similar effects.

Loss of Baiap2l2 destabilizes the transducing stereocilia of cochlear hair cells and leads to deafness.

Key points Mechanoelectrical transduction at auditory hair cells requires highly specialized stereociliary bundles that project from their apical surface, forming a characteristic graded ‘staircase’ structure.The morphogenesis and maintenance of these stereociliary bundles is a tightly regulated process requiring the involvement of several actin‐binding proteins, many of which are still unidentified.We identify a new stereociliary protein, the I‐BAR protein BAIAP2L2, which localizes to the tips of the shorter transducing stereocilia in both inner and outer hair cells (IHCs and OHCs).We find that Baiap2l2 deficient mice lose their second and third rows of stereocilia, their mechanoelectrical transducer current, and develop progressive hearing loss, becoming deaf by 8 months of age.We demonstrate that BAIAP2L2 localization to stereocilia tips is dependent on the motor protein MYO15A and its cargo EPS8.We propose that BAIAP2L2 is a new key protein required for the maintenance of the transducing stereocilia in mature cochlear hair cells. The transduction of sound waves into electrical signals depends upon mechanosensitive stereociliary bundles that project from the apical surface of hair cells within the cochlea. The height and width of these actin‐based stereocilia is tightly regulated throughout life to establish and maintain their characteristic staircase‐like structure, which is essential for normal mechanoelectrical transduction. Here, we show that BAIAP2L2, a member of the I‐BAR protein family, is a newly identified hair bundle protein that is localized to the tips of the shorter rows of transducing stereocilia in mouse cochlear hair cells. BAIAP2L2 was detected by immunohistochemistry from postnatal day 2.5 (P2.5) throughout adulthood. In Baiap2l2 deficient mice, outer hair cells (OHCs), but not inner hair cells (IHCs), began to lose their third row of stereocilia and showed a reduction in the size of the mechanoelectrical transducer current from just after P9. Over the following post‐hearing weeks, the ordered staircase structure of the bundle progressively deteriorates, such that, by 8 months of age, both OHCs and IHCs of Baiap2l2 deficient mice have lost most of the second and third rows of stereocilia and become deaf. We also found that BAIAP2L2 interacts with other key stereociliary proteins involved in normal hair bundle morphogenesis, such as CDC42, RAC1, EPS8 and ESPNL. Furthermore, we show that BAIAP2L2 localization to the stereocilia tips depends on the motor protein MYO15A and its cargo EPS8. We propose that BAIAP2L2 is key to maintenance of the normal actin structure of the transducing stereocilia in mature mouse cochlear hair cells.

Biophysical and morphological changes in inner hair cells and their efferent innervation in the ageing mouse cochlea.

Age-related hearing loss is a progressive hearing loss involving environmental and genetic factors, leading to a decrease in hearing sensitivity, threshold and speech discrimination. We compared age-related changes in inner hair cells (IHCs) between four mouse strains with different levels of progressive hearing loss. The surface area of apical coil IHCs (9-12kHz cochlear region) decreases by about 30-40% with age. The number of BK channels progressively decreases with age in the IHCs from most mouse strains, but the basolateral membrane current profile remains unchanged. The mechanoelectrical transducer current is smaller in mice harbouring the hypomorphic Cdh23 allele Cdh23ahl (C57BL/6J; C57BL/6NTac), but not in Cdh23-repaired mice (C57BL/6NTacCdh23+ ), indicating that it could contribute to the different progression of hearing loss among mouse strains. The degree of efferent rewiring onto aged IHCs, most likely coming from the lateral olivocochlea fibres, was correlated with hearing loss in the different mouse strains. Inner hair cells (IHCs) are the primary sensory receptors of the mammalian cochlea, transducing acoustic information into electrical signals that are relayed to the afferent neurons. Functional changes in IHCs are a potential cause of age-related hearing loss. Here, we have investigated the functional characteristics of IHCs from early-onset hearing loss mice harbouring the allele Cdh23ahl (C57BL/6J and C57BL/6NTac), from late-onset hearing loss mice (C3H/HeJ), and from mice corrected for the Cdh23ahl mutation (C57BL/6NTacCdh23+ ) with an intermediate hearing phenotype. There was no significant loss of IHCs in the 9-12kHz cochlear region up to at least 15months of age, but their surface area decreased progressively by 30-40% starting from ∼6months of age. Although the size of the BK current decreased with age, IHCs retained a normal KCNQ4 current and resting membrane potential. These basolateral membrane changes were most severe for C57BL/6J and C57BL/6NTac, less so for C57BL/6NTacCdh23+ and minimal or absent in C3H/HeJ mice. We also found that lateral olivocochlear (LOC) efferent fibres re-form functional axon-somatic connections with aged IHCs, but this was seen only sporadically in C3H/HeJ mice. The efferent post-synaptic SK2 channels appear prior to the establishment of the efferent contacts, suggesting that IHCs may play a direct role in re-establishing the LOC-IHC synapses. Finally, we showed that the size of the mechanoelectrical transducer (MET) current from IHCs decreased significantly with age in mice harbouring the Cdh23ahl allele but not in C57BL/6NTacCdh23+ mice, indicating that the MET apparatus directly contributes to the progression of age-related hearing loss.

Age-related changes in the biophysical and morphological characteristics of mouse cochlear outer hair cells.

Age-related hearing loss (ARHL) is a very heterogeneous disease, resulting from cellular senescence, genetic predisposition and environmental factors (e.g. noise exposure). Currently, we know very little about age-related changes occurring in the auditory sensory cells, including those associated with the outer hair cells (OHCs). Using different mouse strains, we show that OHCs undergo several morphological and biophysical changes in the ageing cochlea. Ageing OHCs also exhibited the progressive loss of afferent and efferent synapses. We also provide evidence that the size of the mechanoelectrical transducer current is reduced in ageing OHCs, highlighting its possible contribution in cochlear ageing. Outer hair cells (OHCs) are electromotile sensory receptors that provide sound amplification within the mammalian cochlea. Although OHCs appear susceptible to ageing, the progression of the pathophysiological changes in these cells is still poorly understood. By using mouse strains with a different progression of hearing loss (C57BL/6J, C57BL/6NTac, C57BL/6NTacCdh23+ , C3H/HeJ), we have identified morphological, physiological and molecular changes in ageing OHCs (9-12kHz cochlear region). We show that by 6months of age, OHCs from all strains underwent a reduction in surface area, which was not a sign of degeneration. Although the ageing OHCs retained a normal basolateral membrane protein profile, they showed a reduction in the size of the K+ current and non-linear capacitance, a readout of prestin-dependent electromotility. Despite these changes, OHCs have a normal Vm and retain the ability to amplify sound, as distortion product otoacoustic emission thresholds were not affected in aged, good-hearing mice (C3H/HeJ, C57BL/6NTacCdh23+ ). The loss of afferent synapses was present in all strains at 15months. The number of efferent synapses per OHCs, defined as postsynaptic SK2 puncta, was reduced in aged OHCs of all strains apart from C3H mice. Several of the identified changes occurred in aged OHCs from all mouse strains, thus representing a general trait in the pathophysiological progression of age-related hearing loss, possibly aimed at preserving functionality. We have also shown that the mechanoelectrical transduction (MET) current from OHCs of mice harbouring the Cdh23ahl allele is reduced with age, highlighting the possibility that changes in the MET apparatus could play a role in cochlear ageing.

The contribution of TMC1 to adaptation of mechanoelectrical transduction channels in cochlear outer hair cells.

Key points Hair cell mechanoelectrical transducer channels are opened by deflections of the hair bundle about a resting position set by incompletely understood adaptation mechanisms.We used three characteristics to define adaptation in hair cell mutants of transmembrane channel‐like proteins, TMC1 and TMC2, which are considered to be channel constituents.The results obtained demonstrate that the three characteristics are not equivalent, and raise doubts about simple models in which intracellular Ca2+ regulates adaptation.Adaptation is faster and more effective in TMC1‐containing than in TMC2‐containing transducer channels. This result ties adaptation to the channel complex, and suggests that TMC1 is a better isoform for use in cochlear hair cells.We describe a TMC1 point mutation, D569N, that reduces the resting open probability and Ca2+ permeability of the transducer channels, comprising properties that may contribute to the deafness phenotype. Recordings of mechanoelectrical transducer (MET) currents in cochlear hair cells were made in mice with mutations of transmembrane channel‐like (TMC) protein to examine the effects on fast transducer adaptation. Adaptation was faster and more complete in Tmc2–/– than in Tmc1–/–, although this disparity was not explained by differences in Ca2+ permeability or Ca2+ influx between the two isoforms, with TMC2 having the larger permeability. We made a mouse mutation, Tmc1 p.D569N, homologous to a human DFNA36 deafness mutation, which also had MET channels with lower Ca2+‐permeability but showed better fast adaptation than wild‐type Tmc1+/+ channels. Consistent with the more effective adaptation in Tmc1 p.D569N, the resting probability of MET channel opening was smaller. The three TMC variants studied have comparable single‐channel conductances, although the lack of correlation between channel Ca2+ permeability and adaptation opposes the hypothesis that adaptation is controlled simply by Ca2+ influx through the channels. During the first postnatal week of mouse development, the MET currents amplitude grew, and transducer adaptation became faster and more effective. We attribute changes in adaptation partly to a developmental switch from TMC2‐ to TMC1‐ containing channels and partly to an increase in channel expression. More complete and faster adaptation, coupled with larger MET currents, may account for the sole use of TMC1 in the adult cochlear hair cells.

A Tmc1 mutation reduces calcium permeability and expression of mechanoelectrical transduction channels in cochlear hair cells

Mechanoelectrical transducer (MET) currents were recorded from cochlear hair cells in mice with mutations of transmembrane channel-like protein TMC1 to study the effects on MET channel properties. We characterized a Tmc1 mouse with a single-amino-acid mutation (D569N), homologous to a dominant human deafness mutation. Measurements were made in both Tmc2 wild-type and Tmc2 knockout mice. By 30 d, Tmc1 pD569N heterozygote mice were profoundly deaf, and there was substantial loss of outer hair cells (OHCs). MET current in OHCs of Tmc1 pD569N mutants developed over the first neonatal week to attain a maximum amplitude one-third the size of that in Tmc1 wild-type mice, similar at apex and base, and lacking the tonotopic size gradient seen in wild type. The MET-channel Ca2+ permeability was reduced 3-fold in Tmc1 pD569N homozygotes, intermediate deficits being seen in heterozygotes. Reduced Ca2+ permeability resembled that of the Tmc1 pM412K Beethoven mutant, a previously studied semidominant mouse mutation. The MET channel unitary conductance, assayed by single-channel recordings and by measurements of current noise, was unaffected in mutant apical OHCs. We show that, in contrast to the Tmc1 M412K mutant, there was reduced expression of the TMC1 D569N channel at the transduction site assessed by immunolabeling, despite the persistence of tip links. The reduction in MET channel Ca2+ permeability seen in both mutants may be the proximate cause of hair-cell apoptosis, but changes in bundle shape and protein expression in Tmc1 D569N suggest another role for TMC1 apart from forming the channel.

Nelson's notch in the rate-level functions of auditory-nerve fibers might be caused by PIEZO2-mediated reverse-polarity currents in hair cells

Half a century ago, Nelson Kiang and colleagues observed that some spike rate versus stimulus level functions of auditory-nerve-fiber responses to tones are nonmonotonic, exhibiting a sharp notch (“Nelson's notch”) at high stimulus levels. The notch is associated with an abrupt change in the phase of the response by ∼180°. To account for these findings, Kiang proposed a “two-factor cancellation hypothesis” according to which excitation of an auditory-nerve fiber is produced by the sum of two phase-locked factors with opposite phase and with different dependencies on stimulus level, cancelling each other at stimulus levels within the notch. The first factor, which dominates at lower stimulus levels, is likely based on the mechanoelectrical transducer current through mechanoelectrical transducer channels in the tips of the inner-hair-cell stereocilia. The identity of the second factor, which dominates at high stimulus levels, has remained elusive. Here, we hypothesize that the second factor is based on the “anomalous” or “reverse-polarity” currents entering inner hair cells via a second type of mechanosensitive channel, PIEZO2, recently identified in the apical surface of hair cells rather than the tips of their stereocilia.

KCNQ1 rescues TMC1 plasma membrane expression but not mechanosensitive channel activity.

Transmembrane channel-like protein isoform 1 (TMC1) is essential for the generation of mechano-electrical transducer currents in hair cells of the inner ear. TMC1 disruption causes hair cell degeneration and deafness in mice and humans. Although thought to be expressed at the cell surface in vivo, TMC1 remains in the endoplasmic reticulum when heterologously expressed in standard cell lines, precluding determination of its roles in mechanosensing and pore formation. Here, we report that the KCNQ1 Kv channel forms complexes with TMC1 and rescues its surface expression when coexpressed in Chinese Hamster Ovary cells. TMC1 rescue is specific for KCNQ1 within the KCNQ family, is prevented by a KCNQ1 trafficking-deficient mutation, and is influenced by KCNE β subunits and inhibition of KCNQ1 endocytosis. TMC1 lowers KCNQ1 and KCNQ1-KCNE1 K+ currents, and despite the surface expression, it does not detectably respond to mechanical stimulation or high salt. We conclude that TMC1 is not intrinsically mechano- or osmosensitive but has the capacity for cell surface expression, and requires partner protein(s) for surface expression and mechanosensitivity. We suggest that KCNQ1, expression of which is not thought to overlap with TMC1 in hair cells, is a proxy partner bearing structural elements or a sequence motif reminiscent of a true in vivo TMC1 hair cell partner. Discovery of the first reported strategy to rescue TMC1 surface expression should aid future studies of the TMC1 function and native partners.

Loss of ARHGEF6 Causes Hair Cell Stereocilia Deficits and Hearing Loss in Mice.

ARHGEF6 belongs to the family of guanine nucleotide exchange factors (GEFs) for Rho GTPases, and it specifically activates Rho GTPases CDC42 and RAC1. Arhgef6 is the X-linked intellectual disability gene also known as XLID46, and clinical features of patients carrying Arhgef6 mutations include intellectual disability and, in some cases, sensorineural hearing loss. Rho GTPases act as molecular switches in many cellular processes. Their activities are regulated by binding or hydrolysis of GTP, which is facilitated by GEFs and GTPase-activating proteins, respectively. RAC1 and CDC42 have been shown to play important roles in hair cell (HC) stereocilia development. However, the role of ARHGEF6 in inner ear development and hearing function has not yet been investigated. Here, we found that ARHGEF6 is expressed in mouse cochlear HCs, including the HC stereocilia. We established Arhgef6 knockdown mice using the clustered regularly interspaced short palindromic repeat-associated Cas9 nuclease (CRISPR-Cas9) genome editing technique. We showed that ARHGEF6 was indispensable for the maintenance of outer hair cell (OHC) stereocilia, and loss of ARHGEF6 in mice caused HC stereocilia deficits that eventually led to progressive HC loss and hearing loss. However, the loss of ARHGEF6 did not affect the synapse density and did not affect the mechanoelectrical transduction currents in OHCs at postnatal day 3. At the molecular level, the levels of active CDC42 and RAC1 were dramatically decreased in the Arhgef6 knockdown mice, suggesting that ARHGEF6 regulates stereocilia maintenance through RAC1/CDC42.

Mechanotransduction is required for establishing and maintaining mature inner hair cells and regulating efferent innervation

In the adult auditory organ, mechanoelectrical transducer (MET) channels are essential for transducing acoustic stimuli into electrical signals. In the absence of incoming sound, a fraction of the MET channels on top of the sensory hair cells are open, resulting in a sustained depolarizing current. By genetically manipulating the in vivo expression of molecular components of the MET apparatus, we show that during pre-hearing stages the MET current is essential for establishing the electrophysiological properties of mature inner hair cells (IHCs). If the MET current is abolished in adult IHCs, they revert into cells showing electrical and morphological features characteristic of pre-hearing IHCs, including the re-establishment of cholinergic efferent innervation. The MET current is thus critical for the maintenance of the functional properties of adult IHCs, implying a degree of plasticity in the mature auditory system in response to the absence of normal transduction of acoustic signals.

Loss of CIB2 Causes Profound Hearing Loss and Abolishes Mechanoelectrical Transduction in Mice

Calcium and integrin-binding protein 2 (CIB2) belongs to a protein family with four known members, CIB1 through CIB4, which are characterized by multiple calcium-binding EF-hand domains. Among the family members, the Cib1 and Cib2 genes are expressed in mouse cochlear hair cells, and mutations in the human CIB2 gene have been associated with nonsyndromic deafness DFNB48 and syndromic deafness USH1J. To further explore the function of CIB1 and CIB2 in hearing, we established Cib1 and Cib2 knockout mice using the clustered regularly interspaced short palindromic repeat (CRISPR)-associated Cas9 nuclease (CRISPR/Cas9) genome editing technique. We found that loss of CIB1 protein does not affect auditory function, whereas loss of CIB2 protein causes profound hearing loss in mice. Further investigation revealed that hair cell stereocilia development is affected in Cib2 knockout mice. Noticeably, loss of CIB2 abolishes mechanoelectrical transduction (MET) currents in auditory hair cells. In conclusion, we show here that although both CIB1 and CIB2 are readily detected in the cochlea, only loss of CIB2 results in profound hearing loss, and that CIB2 is essential for auditory hair cell MET.

CIB2, defective in isolated deafness, is key for auditory hair cell mechanotransduction and survival

Defects of CIB2, calcium‐ and integrin‐binding protein 2, have been reported to cause isolated deafness, DFNB48 and Usher syndrome type‐IJ, characterized by congenital profound deafness, balance defects and blindness. We report here two new nonsense mutations (pGln12* and pTyr110*) in CIB2 patients displaying nonsyndromic profound hearing loss, with no evidence of vestibular or retinal dysfunction. Also, the generated CIB2−/− mice display an early onset profound deafness and have normal balance and retinal functions. In these mice, the mechanoelectrical transduction currents are totally abolished in the auditory hair cells, whilst they remain unchanged in the vestibular hair cells. The hair bundle morphological abnormalities of CIB2−/− mice, unlike those of mice defective for the other five known USH1 proteins, begin only after birth and lead to regression of the stereocilia and rapid hair‐cell death. This essential role of CIB2 in mechanotransduction and cell survival that, we show, is restricted to the cochlea, probably accounts for the presence in CIB2−/− mice and CIB2 patients, unlike in Usher syndrome, of isolated hearing loss without balance and vision deficits.

A connexin30 mutation rescues hearing and reveals roles for gap junctions in cochlear amplification and micromechanics

Accelerated age-related hearing loss disrupts high-frequency hearing in inbred CD-1 mice. The p.Ala88Val (A88V) mutation in the gene coding for the gap-junction protein connexin30 (Cx30) protects the cochlear basal turn of adult CD-1Cx30A88V/A88V mice from degeneration and rescues hearing. Here we report that the passive compliance of the cochlear partition and active frequency tuning of the basilar membrane are enhanced in the cochleae of CD-1Cx30A88V/A88V compared to CBA/J mice with sensitive high-frequency hearing, suggesting that gap junctions contribute to passive cochlear mechanics and energy distribution in the active cochlea. Surprisingly, the endocochlear potential that drives mechanoelectrical transduction currents in outer hair cells and hence cochlear amplification is greatly reduced in CD-1Cx30A88V/A88V mice. Yet, the saturating amplitudes of cochlear microphonic potentials in CD-1Cx30A88V/A88V and CBA/J mice are comparable. Although not conclusive, these results are compatible with the proposal that transmembrane potentials, determined mainly by extracellular potentials, drive somatic electromotility of outer hair cells.

BODIPY-Conjugated Xyloside Primes Fluorescent Glycosaminoglycans in the Inner Ear of Opsanus tau

We report on a new xyloside conjugated to BODIPY, BX and its utility to prime fluorescent glycosaminoglycans (BX-GAGs) within the inner ear in vivo. When BX is administered directly into the endolymphatic space of the oyster toadfish (Opsanus tau) inner ear, fluorescent BX-GAGs are primed and become visible in the sensory epithelia of the semicircular canals, utricle, and saccule. Confocal and 2-photon microscopy of vestibular organs fixed 4h following BX treatment, reveal BX-GAGs constituting glycocalyces that envelop hair cell kinocilium, nerve fibers, and capillaries. In the presence of GAG-specific enzymes, the BX-GAG signals are diminished, suggesting that chondroitin sulfates are the primary GAGs primed by BX. Results are consistent with similar click-xylosides in CHO cell lines, where the xyloside enters the Golgi and preferentially initiates chondroitin sulfate B production. Introduction of BX produces a temporary block of hair cell mechanoelectrical transduction (MET) currents in the crista, reduction in background discharge rate of afferent neurons, and a reduction in sensitivity to physiological stimulation. A six-degree-of-freedom pharmacokinetic mathematical model has been applied to interpret the time course and spatial distribution of BX and BX-GAGs. Results demonstrate a new optical approach to study GAG biology in the inner ear, for tracking synthesis and localization in real time.