Spontaneous Otoacoustic Emissions in TectaY1870C/+ Mice Reflect Changes in Cochlear Amplification and How It Is Controlled by the Tectorial Membrane.

Spontaneous otoacoustic emissions (SOAEs) recorded from the ear canal in the absence of sound reflect cochlear amplification, an outer hair cell (OHC) process required for the extraordinary sensitivity and frequency selectivity of mammalian hearing. Although wild-type mice rarely emit, those with mutations that influence the tectorial membrane (TM) show an incidence of SOAEs similar to that in humans. In this report, we characterized mice with a missense mutation in Tecta, a gene required for the formation of the striated-sheet matrix within the core of the TM. Mice heterozygous for the Y1870C mutation (TectaY1870C/+ ) are prolific emitters, despite a moderate hearing loss. Additionally, Kimura's membrane, into which the OHC stereocilia insert, separates from the main body of the TM, except at apical cochlear locations. Multimodal SOAEs are also observed in TectaY1870C/+ mice where energy is present at frequencies that are integer multiples of a lower-frequency SOAE (the primary). Second-harmonic SOAEs, at twice the frequency of a lower-frequency primary, are the most frequently observed. These secondary SOAEs are found in spatial regions where stimulus-evoked OAEs are small or in the noise floor. Introduction of high-level suppressors just above the primary SOAE frequency reduce or eliminate both primary and second-harmonic SOAEs. In contrast, second-harmonic SOAEs are not affected by suppressors, either above or below the second-harmonic SOAE frequency, even when they are much larger in amplitude. Hence, second-harmonic SOAEs do not appear to be spatially separated from their primaries, a finding that has implications for cochlear mechanics and the consequences of changes to TM structure.

Increased Spontaneous Otoacoustic Emissions in Mice with a Detached Tectorial Membrane.

Mutations in genes encoding tectorial membrane (TM) proteins are a significant cause of human hereditary hearing loss (Hildebrand et al. 2011), and several mouse models have been developed to study the functional significance of this accessory structure in the mammalian cochlea. In this study, we use otoacoustic emissions (OAE), signals obtained from the ear canal that provide a measure of cochlear function, to characterize a mouse in which the TM is detached from the spiral limbus due to an absence of otoancorin (Otoa, Lukashkin et al. 2012). Our results demonstrate that spontaneous emissions (SOAE), sounds produced in the cochlea without stimulation, increase dramatically in mice with detached TMs even though their hearing sensitivity is reduced. This behavior is unusual because wild-type (WT) controls are rarely spontaneous emitters. SOAEs in mice lacking Otoa predominate around 7 kHz, which is much lower than in either WT animals when they generate SOAEs or in mutant mice in which the TM protein Ceacam16 is absent (Cheatham et al. 2014). Although both mutants lack Hensen's stripe, loss of this TM feature is only observed in regions coding frequencies greater than ~15 kHz in WT mice so its loss cannot explain the low-frequency, de novo SOAEs observed in mice lacking Otoa. The fact that ~80 % of mice lacking Otoa produce SOAEs even when they generate smaller distortion product OAEs suggests that the active process is still functioning in these mutants but the system(s) involved have become less stable due to alterations in TM structure.

Prestin-Dependence of Outer Hair Cell Survival and Partial Rescue of Outer Hair Cell Loss in PrestinV499G/Y501H Knockin Mice.

A knockin (KI) mouse expressing mutated prestinV499G/Y501H (499 prestin) was created to study cochlear amplification. Recordings from isolated outer hair cells (OHC) in this mutant showed vastly reduced electromotility and, as a consequence, reduced hearing sensitivity. Although 499 prestin OHCs were normal in stiffness and longer than OHCs lacking prestin, accelerated OHC death was unexpectedly observed relative to that documented in prestin knockout (KO) mice. These observations imply an additional role of prestin in OHC maintenance besides its known requirement for mammalian cochlear amplification. In order to gain mechanistic insights into prestin-associated OHC loss, we implemented several interventions to improve survival. First, 499 prestin KI's were backcrossed to Bak KO mice, which lack the mitochondrial pro-apoptotic gene Bak. Because oxidative stress is implicated in OHC death, another group of 499 prestin KI mice was fed the antioxidant diet, Protandim. 499 KI mice were also backcrossed onto the FVB murine strain, which retains excellent high-frequency hearing well into adulthood, to reduce the compounding effect of age-related hearing loss associated with the original 499 prestin KIs. Finally, a compound heterozygous (chet) mouse expressing one copy of 499 prestin and one copy of KO prestin was also created to reduce quantities of 499 prestin protein. Results show reduction in OHC death in chets, and in 499 prestin KIs on the FVB background, but only a slight improvement in OHC survival for mice receiving Protandim. We also report that improved OHC survival in 499 prestin KIs had little effect on hearing phenotype, reaffirming the original contention about the essential role of prestin's motor function in cochlear amplification.

Loss of the tectorial membrane protein CEACAM16 enhances spontaneous, stimulus-frequency, and transiently evoked otoacoustic emissions.

α-Tectorin (TECTA), ß-tectorin (TECTB), and carcinoembryonic antigen-related cell adhesion molecule 16 (CEACAM) are secreted glycoproteins that are present in the tectorial membrane (TM), an extracellular structure overlying the hearing organ of the inner ear, the organ of Corti. Previous studies have shown that TECTA and TECTB are both required for formation of the striated-sheet matrix within which collagen fibrils of the TM are imbedded and that CEACAM16 interacts with TECTA. To learn more about the structural and functional significance of CEACAM16, we created a Ceacam16-null mutant mouse. In the absence of CEACAM16, TECTB levels are reduced, a clearly defined striated-sheet matrix does not develop, and Hensen's stripe, a prominent feature in the basal two-thirds of the TM in WT mice, is absent. CEACAM16 is also shown to interact with TECTB, indicating that it may stabilize interactions between TECTA and TECTB. Although brain-stem evoked responses and distortion product otoacoustic emissions are, for most frequencies, normal in young mice lacking CEACAM16, stimulus-frequency and transiently evoked emissions are larger. We also observed spontaneous otoacoustic emissions (SOAEs) in 70% of the homozygous mice. This incidence is remarkable considering that <3% of WT controls have SOAEs. The predominance of SOAEs >15 kHz correlates with the loss of Hensen's stripe. Results from mice lacking CEACAM16 are consistent with the idea that the organ of Corti evolved to maximize the gain of the cochlear amplifier while preventing large oscillations. Changes in TM structure appear to influence the balance between energy generation and dissipation such that the system becomes unstable.

Functional regulation of the SLC26-family protein prestin by calcium/calmodulin.

The solute carrier gene family 26 (SLC26) encodes membrane proteins with diverse physiological roles but with the common feature of halide involvement. Here, we present bioinformatic and biochemical evidence that SLC26 proteins have intrinsically disordered regions (IDRs) in their C-terminal domains and that these regions contain calmodulin (CaM) binding sites. The veracity of these predictions and the functional consequences of CaM binding were examined in prestin, SLC26A5, as a model for the SLC26 family and as one of the most investigated and best understood members. We found that CaM binds directly to the IDR in the C-terminal domain of prestin in a calcium-obligate manner. Using both isolated murine outer hair cells (OHCs) and a heterologous expression system, we also found that this calcium-obligate CaM binding shifts the operating point of the protein to more hyperpolarized potentials with consequent alteration of the function of the prestin. Because calcium is the main intracellular second messenger used by the efferent medial olivocochlear (MOC) pathway of the auditory system and CaM is abundant in OHCs, the CaM-prestin interaction may be involved in the MOC-mediated modulation of cochlear amplification. However, this regulatory mechanism is not likely to be restricted to cochlear OHCs, in light of both clear bioinformatic evidence and the fact that calcium and CaM are ubiquitous intracellular second messengers used by virtually all cell types. Hence, the calcium/CaM-dependent regulatory mechanism described herein is likely applicable to most, if not all, SLC26 paralogs.

Marshalin, a microtubule minus-end binding protein, regulates cytoskeletal structure in the organ of Corti.

Dramatic structural changes in microtubules (MT) and the assembly of complicated intercellular connections are seen during the development of the cellular matrix of the sense organ for hearing, the organ of Corti. This report examines the expression of marshalin, a minus-end binding protein, during this process of cochlear development. We discovered that marshalin is abundantly expressed in both sensory hair cells and supporting cells. In the adult, prominent marshalin expression is observed in the cuticular plates of hair cells and in the noncentrosomal MT organization centers (MTOC) of Deiters' and pillar cells. Based upon differences in marshalin expression patterns seen in the organ of Corti, we identified eight isoforms ranging from 863 to 1280 amino acids. mRNAs/proteins associated with marshalin's isoforms are detected at different times during development. These isoforms carry various protein-protein interacting domains, including coiled-coil (CC), calponin homology (CH), proline-rich (PR), and MT-binding domains, referred to as CKK. We, therefore, examined membranous organelles and structural changes in the cytoskeleton induced by expressing two of these marshalin isoforms in vitro. Long forms containing CC and PR domains induce thick, spindle-shaped bundles, whereas short isoforms lacking CC and PR induce more slender variants that develop into densely woven networks. Together, these data suggest that marshalin is closely associated with noncentrosomal MTOCs, and may be involved in MT bundle formation in supporting cells. As a scaffolding protein with multiple isoforms, marshalin is capable of modifying cytoskeletal networks, and consequently organelle positioning, through interactions with various protein partners present in different cells.

Pixels as ROIs (PAR): a less-biased and statistically powerful approach for gleaning functional information from image stacks.

Especially in the last decade or so, there have been dramatic advances in fluorescence-based imaging methods designed to measure a multitude of functions in living cells. Despite this, many of the methods used to analyze the resulting images are limited. Perhaps the most common mode of analysis is the choice of regions of interest (ROIs), followed by quantification of the signal contained therein in comparison with another "control" ROI. While this method has several advantages, such as flexibility and capitalization on the power of human visual recognition capabilities, it has the drawbacks of potential subjectivity and lack of precisely defined criteria for ROI selection. This can lead to analyses which are less precise or accurate than the data might allow for, and generally a regrettable loss of information. Herein, we explore the possibility of abandoning the use of conventional ROIs, and instead propose treating individual pixels as ROIs, such that all information can be extracted systematically with the various statistical cutoffs we discuss. As a test case for this approach, we monitored intracellular pH in cells transfected with the chloride/bicarbonate transporter slc26a3 using the ratiometric dye SNARF-5F under various conditions. We performed a parallel analysis using two different levels of stringency in conventional ROI analysis as well as the pixels-as-ROIs (PAR) approach, and found that pH differences between control and transfected cells were accentuated by ~50-100% by using the PAR approach. We therefore consider this approach worthy of adoption, especially in cases in which higher accuracy and precision are required.

The V499G/Y501H mutation impairs fast motor kinetics of prestin and has significance for defining functional independence of individual prestin subunits.

Outer hair cells (OHCs) are a mammalian innovation for mechanically amplifying sound energy to overcome the viscous damping of the cochlear partition. Although the voltage-dependent OHC membrane motor, prestin, has been demonstrated to be essential for mammalian cochlear amplification, the molecular mechanism by which prestin converts electrical energy into mechanical displacement/force remains elusive. Identifying mutations that alter the motor function of prestin provides vital information for unraveling the energy transduction mechanism of prestin. We show that the V499G/Y501H mutation does not deprive prestin of its voltage-induced motor activity, but it does significantly impair the fast motor kinetics and voltage operating range. Furthermore, mutagenesis studies suggest that Val-499 is the primary site responsible for these changes. We also show that V499G/Y501H prestin forms heteromers with wild-type prestin and that the fast motor kinetics of wild-type prestin is not affected by heteromer formation with V499G/Y501H prestin. These results suggest that prestin subunits are individually functional within a given multimer.

Dissecting the electromechanical coupling mechanism of the motor-protein prestin.

Prestin, which is a member of the solute carrier 26 anion transporter family (SLC26A5), is a voltage-dependent membrane-based motor protein that confers electromotility on mammalian cochlear outer hair cells (OHCs).1 OHCs are a mammalian innovation, their presence2 and their endowment with functional prestin is essential for normal hearing of mammals.3 In order to clarify the molecular mechanism underlying the voltage-dependent motility of prestin, precise description of the relation between voltage-induced prestin-associated charge movement and the resulting cell displacement is essential. By simultaneously measuring voltage-dependent charge movement, which is manifested in the nonlinear capacitance (NLC) of the cell membrane, and voltage-induced OHC displacement, we provided compelling experimental evidence that prestin-associated charge movement and the resulting electromotility are fully coupled, and that prestin has at least two voltage-dependent conformational transition steps. These findings provide a basis for understanding the molecular mechanism of prestin. Here we discuss the relevance of our finding in the elucidation of the voltage-dependent motor mechanism of prestin, and speculate about possible voltage sensing mechanisms of the molecule.

Carcinoembryonic antigen-related cell adhesion molecule 16 interacts with alpha-tectorin and is mutated in autosomal dominant hearing loss (DFNA4).

We report on a secreted protein found in mammalian cochlear outer hair cells (OHC) that is a member of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family of adhesion proteins. Ceacam16 mRNA is expressed in OHC, and its protein product localizes to the tips of the tallest stereocilia and the tectorial membrane (TM). This specific localization suggests a role in maintaining the integrity of the TM as well as in the connection between the OHC stereocilia and TM, a linkage essential for mechanical amplification. In agreement with this role, CEACAM16 colocalizes and coimmunoprecipitates with the TM protein α-tectorin. In addition, we show that mutation of CEACAM16 leads to autosomal dominant nonsyndromic deafness (ADNSHL) at the autosomal dominant hearing loss (DFNA4) locus. In aggregate, these data identify CEACAM16 as an α-tectorin-interacting protein that concentrates at the point of attachment of the TM to the stereocilia and, when mutated, results in ADNSHL at the DFNA4 locus.

Using the cochlear microphonic as a tool to evaluate cochlear function in mouse models of hearing.

The cochlear microphonic (CM) can be a useful analytical tool, but many investigators may not be fully familiar with its unique properties to interpret it accurately in mouse models of hearing. The purpose of this report is to develop a model for generation of the CM in wild-type (WT) and prestin knockout mice. Data and modeling results indicate that in the majority of cases, the CM is a passive response, and in the absence of outer hair cell (OHC) damage, mice lacking amplification are expected to generate WT levels of CM for inputs less than approximately 30 kHz. Hence, this cochlear potential is not a useful metric to estimate changes in amplifier gain. This modeling analysis may explain much of the paradoxical data in the literature. For example, various manipulations, including the application of salicylate and activation of the crossed olivocochlear bundle, reduce the compound action potential but increase or do not change the CM. Based on this current evaluation, CM measurements are consistent with early descriptions where this AC cochlear potential is dominated by basal OHCs, when recorded at the round window.

Evidence that prestin has at least two voltage-dependent steps.

Prestin is a voltage-dependent membrane-spanning motor protein that confers electromotility on mammalian cochlear outer hair cells, which is essential for normal hearing of mammals. Voltage-induced charge movement in the prestin molecule is converted into mechanical work; however, little is known about the molecular mechanism of this process. For understanding the electromechanical coupling mechanism of prestin, we simultaneously measured voltage-dependent charge movement and electromotility under conditions in which the magnitudes of both charge movement and electromotility are gradually manipulated by the prestin inhibitor, salicylate. We show that the observed relationships of the charge movement and the physical displacement (q-d relations) are well represented by a three-state Boltzmann model but not by a two-state model or its previously proposed variant. Here, we suggest a molecular mechanism of prestin with at least two voltage-dependent conformational transition steps having distinct electromechanical coupling efficiencies.

Interaction between the motor protein prestin and the transporter protein VAPA.

Prestin is the motor protein responsible for cochlear outer hair cell (OHC) somatic electromotility. Eliminating this abundant basolateral membrane protein not only causes loss of frequency selectivity and hearing sensitivity, but also leads to OHC death. A membrane-based yeast two-hybrid approach was used to screen an OHC-enriched cDNA (complementary Deoxyribonucleic Acid) library in order to identify prestin-associated proteins. Several proteins were recognized as potential prestin partners, including vesicle-associated membrane protein associated protein A (VAPA or VAP-33). VAPA is an integral membrane protein that plays an important role in membrane trafficking, endoplasmic reticulum homeostasis, and the stress-signaling system. The connection between VAPA and prestin was confirmed through co-immunoprecipitation experiments. This new finding prompted the investigation of the interaction between VAPA and prestin in outer hair cells. By comparing VAPA expression between wild-type OHCs and OHCs derived from prestin-knockout mice, we found that VAPA is expressed in OHCs and the quantity of VAPA expressed is related to the presence of prestin. In other words, less VAPA protein is found in OHCs lacking prestin. Thus, prestin appears to modify the expression of VAPA protein in OHCs. Intriguingly, more prestin protein appears at the plasma membrane when VAPA is co-expressed with prestin. These data suggest that VAPA could be involved in prestin's transportation inside OHCs and may facilitate the targeting of this abundant OHC protein to the plasma membrane.

WITHDRAWN: Feedback in the cochlea.

This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

Interaction between CFTR and prestin (SLC26A5).

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel that is present in a variety of epithelial cell types, and usually expressed in the luminal membrane. In contrast, prestin (SLC26A5) is a voltage-dependent motor protein, which is present in the basolateral membrane of cochlear outer hair cells (OHCs), and plays an important role in the frequency selectivity and sensitivity of mammalian hearing. By using in situ hybridization and immunofluorescence, we found that both mRNA and protein of CFTR are present in OHCs, and that CFTR localizes in both the apical and the lateral membranes. CFTR was not detected in the lateral membrane of inner hair cells (IHCs) or in that of OHCs derived from prestin-knockout mice, i.e., in instances where prestin is not expressed. These results suggest that prestin may interact physically with CFTR in the lateral membrane of OHCs. Immunoprecipitation experiments confirmed a prestin-CFTR interaction. Because chloride is important for prestin function and for the efferent-mediated inhibition of cochlear output, the prestin-directed localization of CFTR to the lateral membrane of OHCs has a potential physiological significance. Aside from its role as a chloride channel, CFTR is known as a regulator of multiple protein functions, including those of the solute carrier family 26 (SLC26). Because prestin is in the SLC26 family, several members of which interact with CFTR, we explored the potential modulatory relationship associated with a direct, physical interaction between prestin and CFTR. Electrophysiological experiments demonstrated that cAMP-activated CFTR is capable of enhancing voltage-dependent charge displacement, a signature of OHC motility, whereas prestin does not affect the chloride conductance of CFTR.

A chimera analysis of prestin knock-out mice.

A chimera is a genetic composite containing a unique mix of cells derived from more than one zygote. This mouse model allows one to learn how cells of contrasting genotype functionally interact in vivo. Here, we investigate the effect that different proportions of prestin-containing outer hair cells (OHC) have on cochlear amplification. To address this issue, we developed a prestin chimeric mouse in which both ROSA26 wild-type (WT) and prestin knock-out (KO) genotypes are present in a single cochlea. The WT ROSA26 mice express a cell marker, allowing one to identify cells originating from the WT genome. Examination of cochlear tissue indicated that prestin chimeric mice demonstrate a mosaic in which mutant and normal OHCs interleave along the cochlear partition, similar to all other chimeric mouse models. The anatomical distribution of prestin-containing OHCs was compared with physiological data including thresholds and tuning curves for the compound action potential (CAP) recorded in anesthetized mice. Analysis of these measures did not reveal mixed phenotypes in which the distribution of prestin-containing OHCs impacted sensitivity and frequency selectivity to different degrees. However, by reducing the number of prestin-containing OHCs, phenotypes intermediate between WT and KO response patterns were obtained. Accordingly, we demonstrate a proportional reduction in sensitivity and in the tip length of CAP tuning curves as the number of OHCs derived from the KO genome increases; i.e., genotype ratio and phenotype are closely related.

EHD4 and CDH23 are interacting partners in cochlear hair cells.

Cadherin 23 (CDH23), a transmembrane protein localized near the tips of hair cell stereocilia in the mammalian inner ear, is important for delivering mechanical signals to the mechano-electric transducer channels. To identify CDH23-interacting proteins, a membrane-based yeast two-hybrid screen of an outer hair cell (OHC) cDNA library was performed. EHD4, a member of the C-terminal EH domain containing a protein family involved in endocytic recycling, was identified as a potential interactor. To confirm the interaction, we first demonstrated the EHD4 mRNA expression in hair cells using in situ hybridization. Next, we showed that EHD4 co-localizes and co-immunoprecipitates with CDH23 in mammalian cells. Interestingly, the co-immunoprecipitation was found to be calcium-sensitive. To investigate the role of EHD4 in hearing, compound action potentials were measured in EHD4 knock-out (KO) mice. Although EHD4 KO mice have normal hearing sensitivity, analysis of mouse cochlear lysates revealed a 2-fold increase in EHD1, but no increase in EHD2 or EHD3, in EHD4 KO cochleae compared with wild type, suggesting that a compensatory increase in EHD1 levels may account for the absence of a hearing defect in EHD4 KO mice. Taken together, these data indicate that EHD4 is a novel CDH23-interacting protein that could regulate CDH23 trafficking/localization in a calcium-sensitive manner.

Identifying components of the hair-cell interactome involved in cochlear amplification.

BACKGROUND: Although outer hair cells (OHCs) play a key role in cochlear amplification, it is not fully understood how they amplify sound signals by more than 100 fold. Two competing or possibly complementary mechanisms, stereocilia-based and somatic electromotility-based amplification, have been considered. Lacking knowledge about the exceptionally rich protein networks in the OHC plasma membrane, as well as related protein-protein interactions, limits our understanding of cochlear function. Therefore, we focused on finding protein partners for two important membrane proteins: Cadherin 23 (cdh23) and prestin. Cdh23 is one of the tip-link proteins involved in transducer function, a key component of mechanoelectrical transduction and stereocilia-based amplification. Prestin is a basolateral membrane protein responsible for OHC somatic electromotility. RESULTS: Using the membrane-based yeast two-hybrid system to screen a newly built cDNA library made predominantly from OHCs, we identified two completely different groups of potential protein partners using prestin and cdh23 as bait. These include both membrane bound and cytoplasmic proteins with 12 being de novo gene products with unknown function(s). In addition, some of these genes are closely associated with deafness loci, implying a potentially important role in hearing. The most abundant prey for prestin (38%) is composed of a group of proteins involved in electron transport, which may play a role in OHC survival. The most abundant group of cdh23 prey (55%) contains calcium-binding domains. Since calcium performs an important role in hair cell mechanoelectrical transduction and amplification, understanding the interactions between cdh23 and calcium-binding proteins should increase our knowledge of hair cell function at the molecular level. CONCLUSION: The results of this study shed light on some protein networks in cochlear hair cells. Not only was a group of de novo genes closely associated with known deafness loci identified, but the data also indicate that the hair cell tip link interacts directly with calcium binding proteins. The OHC motor protein, prestin, also appears to be associated with electron transport proteins. These unanticipated results open potentially fruitful lines of investigation into the molecular basis of cochlear amplification.

Cochlear amplification, outer hair cells and prestin.

Mechanical amplification of acoustic signals is apparently a common feature of vertebrate auditory organs. In non-mammalian vertebrates amplification is produced by stereociliary processes, related to the mechanotransducer channel complex and probably to the phenomenon of fast adaptation. The extended frequency range of the mammalian cochlea has probably co-evolved with a novel hair cell type, the outer hair cell and its constituent membrane protein, prestin. Cylindrical outer hair cells are motile and their somatic length changes are voltage driven and powered by prestin. One of the central outstanding problems in mammalian cochlear neurobiology is the relation between the two amplification processes.