Molecular mechanism of prestin electromotive signal amplification.

Hearing involves two fundamental processes: mechano-electrical transduction and signal amplification. Despite decades of studies, the molecular bases for both remain elusive. Here, we show how prestin, the electromotive molecule of outer hair cells (OHCs) that senses both voltage and membrane tension, mediates signal amplification by coupling conformational changes to alterations in membrane surface area. Cryoelectron microscopy (cryo-EM) structures of human prestin bound with chloride or salicylate at a common "anion site" adopt contracted or expanded states, respectively. Prestin is ensconced within a perimeter of well-ordered lipids, through which it induces dramatic deformation in the membrane and couples protein conformational changes to the bulk membrane. Together with computational studies, we illustrate how the anion site is allosterically coupled to changes in the transmembrane domain cross-sectional area and the surrounding membrane. These studies provide insight into OHC electromotility by providing a structure-based mechanism of the membrane motor prestin.

Prestin's fast motor kinetics is essential for mammalian cochlear amplification.

Prestin (SLC26A5)-mediated voltage-driven elongations and contractions of sensory outer hair cells within the organ of Corti are essential for mammalian cochlear amplification. However, whether this electromotile activity directly contributes on a cycle-by-cycle basis is currently controversial. By restoring motor kinetics in a mouse model expressing a slowed prestin missense variant, this study provides experimental evidence acknowledging the importance of fast motor action to mammalian cochlear amplification. Our results also demonstrate that the point mutation in prestin disrupting anion transport in other proteins of the SLC26 family does not alter cochlear function, suggesting that the potential weak anion transport of prestin is not essential in the mammalian cochlea.

The molecular principles underlying diverse functions of the SLC26 family of proteins.

Mammalian SLC26 proteins are membrane-based anion transporters that belong to the large SLC26/SulP family, and many of their variants are associated with hereditary diseases. Recent structural studies revealed a strikingly similar homodimeric molecular architecture for several SLC26 members, implying a shared molecular principle. Now a new question emerges as to how these structurally similar proteins execute diverse physiological functions. In this study, we sought to identify the common versus distinct molecular mechanism among the SLC26 proteins using both naturally occurring and artificial missense changes introduced to SLC26A4, SLC26A5, and SLC26A9. We found: (i) the basic residue at the anion binding site is essential for both anion antiport of SLC26A4 and motor functions of SLC26A5, and its conversion to a nonpolar residue is crucial but not sufficient for the fast uncoupled anion transport in SLC26A9; (ii) the conserved polar residues in the N- and C-terminal cytosolic domains are likely involved in dynamic hydrogen-bonding networks and are essential for anion antiport of SLC26A4 but not for motor (SLC26A5) and uncoupled anion transport (SLC26A9) functions; (iii) the hydrophobic interaction between each protomer's last transmembrane helices, TM14, is not of functional significance in SLC26A9 but crucial for the functions of SLC26A4 and SLC26A5, likely contributing to optimally orient the axis of the relative movements of the core domain with respect to the gate domains within the cell membrane. These findings advance our understanding of the molecular mechanisms underlying the diverse physiological roles of the SLC26 family of proteins.

Megahertz Sampling of Prestin (SLC26a5) Voltage-Sensor Charge Movements in Outer Hair Cell Membranes Reveals Ultrasonic Activity that May Support Electromotility and Cochlear Amplification.

Charged moieties in the outer hair cell (OHC) membrane motor protein, prestin, are driven by transmembrane voltage to power OHC electromotility (eM) and cochlear amplification (CA), an enhancement of mammalian hearing. Consequently, the speed of prestin's conformational switching constrains its dynamic influence on micromechanics of the cell and the organ of Corti. Corresponding voltage-sensor charge movements in prestin, classically assessed as a voltage-dependent, nonlinear membrane capacitance (NLC), have been used to gauge its frequency response, but have been validly measured only out to 30 kHz. Thus, controversy exists concerning the effectiveness of eM in supporting CA at ultrasonic frequencies where some mammals can hear. Using megahertz sampling of guinea pig (either sex) prestin charge movements, we extend interrogations of NLC into the ultrasonic range (up to 120 kHz) and find an order of magnitude larger response at 80 kHz than previously predicted, indicating that an influence of eM at ultrasonic frequencies is likely, in line with recent in vivo results (Levic et al., 2022). Given wider bandwidth interrogations, we also validate kinetic model predictions of prestin by directly observing its characteristic cut-off frequency under voltage-clamp as the intersection frequency (Fis), near 19 kHz, of the real and imaginary components of complex NLC (cNLC). The frequency response of prestin displacement current noise determined from either the Nyquist relation or stationary measures aligns with this cut-off. We conclude that voltage stimulation accurately assesses the spectral limits of prestin activity, and that voltage-dependent conformational switching is physiologically significant in the ultrasonic range.SIGNIFICANCE STATEMENT The motor protein prestin powers outer hair cell (OHC) electromotility (eM) and cochlear amplification (CA), an enhancement of high-frequency mammalian hearing. The ability of prestin to work at very high frequencies depends on its membrane voltage-driven conformation switching. Using megahertz sampling, we extend measures of prestin charge movement into the ultrasonic range and find response magnitude at 80 kHz an order of magnitude larger than previously estimated, despite confirmation of previous low pass characteristic frequency cut-offs. The frequency response of prestin noise garnered by the admittance-based Nyquist relation or stationary noise measures confirms this characteristic cut-off frequency. Our data indicate that voltage perturbation provides accurate assessment of prestin performance indicating that it can support cochlear amplification into a higher frequency range than previously thought.

The pathogenic roles of the p.R130S prestin variant in DFNB61 hearing loss.

DFNB61 is a recessively inherited nonsyndromic hearing loss caused by mutations in SLC26A5, the gene that encodes the voltage-driven motor protein, prestin. Prestin is abundantly expressed in the auditory outer hair cells that mediate cochlear amplification. Two DFNB61-associated SLC26A5 variants, p.W70X and p.R130S, were identified in patients who are compound heterozygous for these nonsense and missense changes (SLC26A5W70X/R130S ). Our recent study showed that mice homozygous for p.R130S (Slc26a5R130S/R130S ) suffer from hearing loss that is ascribed to significantly reduced motor kinetics of prestin. Given that W70X-prestin is nonfunctional, compound heterozygous Slc26a5R130S/- mice were used as a model for human SLC26A5W70X/R130S . By examining the pathophysiological consequences of p.R130S prestin when it is the sole allele for prestin protein production, we determined that this missense change results in progressive outer hair cell loss in addition to its effects on prestin's motor action. Thus, this study defines the pathogenic roles of p.R130S prestin and identifies a limited time window for potential clinical intervention. KEY POINTS: The voltage-driven motor protein, prestin, is encoded by SLC26A5 and expressed abundantly in cochlear outer hair cells (OHCs). The importance of prestin for normal hearing was demonstrated in mice lacking prestin; however, none of the specific SLC26A5 variants identified to date in human patients has been experimentally demonstrated to be pathogenic. In this study we used both cell lines and a mouse model to define the pathogenic role of compound heterozygous p.W70X (c.209G>A) and p.R130S (c.390A>C) SLC26A5 variants identified in patients with moderate to profound hearing loss. As in patients, mice carrying one copy of p.R130S Slc26a5 showed OHC dysfunction and progressive degeneration, which results in congenital progressive hearing loss. This is the first functional study reporting pathogenic SLC26A5 variants and pointing to the presence of a therapeutic time window for potential clinical interventions targeting the affected OHCs before they are lost.

Differential outcomes of high-fat diet on age-related rescaling of cochlear frequency place coding.

Auditory frequency coding is place-specific, which depends on the mechanical coupling of the basilar membrane-outer hair cell (OHC)-tectorial membrane network. Prestin-based OHC electromotility improves cochlear frequency selectivity and sensitivity. Cochlear amplification determines the frequency coding wherein discrete sound frequencies find a 'best' place along the cochlear length. Loss of OHC is the leading cause of age-related hearing loss (ARHL) and is the most common cause of sensorineural hearing loss and compromised speech perception. Lipid interaction with Prestin impacts OHC function. It has been established that high-fat diet (HFD) is associated with ARHL. To determine whether genetic background and metabolism preserve cochlear frequency place coding, we examined the effect of HFD in C57BL/6J (B6) and CBA/CaJ (CBA) on ARHL.We found a significant rescuing effect on ARHL in aged B6 HFD cohort. Prestin levels and cell sizes were better maintained in the experimental B6-HFD group. We also found that distortion product otoacoustic emission (DPOAE) group delay measurement was preserved, which suggested stable frequency place coding. In contrast, the response to HFD in the CBA cohort was modest with no appreciable benefit to hearing threshold. Notably, group delay was shortened with age along with the control. In addition, the frequency dependent OHC nonlinear capacitance gradient was most pronounced at young age but decreased with age. Cochlear RNA-seq analysis revealed differential TRPV1 expression and lipid homeostasis. Activation of TRPV1 and downregulation of arachidonic acid led to downregulation of inflammatory response in B6 HFD, which protects the cochlea from ARHL. The genetic background and metabolic state-derived changes in OHC morphology and function collectively contribute to a redefined cochlear frequency place coding and improved age-related pitch perception.

Verification of Outer Hair Cell Motor Protein, Prestin, as a Serological Biomarker for Mouse Cochlear Damage.

The motor protein prestin, found in the inner ear's outer hair cells (OHCs), is responsible for high sensitivity and sharp frequency selectivity in mammalian hearing. Some studies have suggested that prestin could be a serological biomarker for cochlear damage, as OHCs are highly vulnerable to damage from various sources. However, the reported data are inconsistent and lack appropriate negative controls. To investigate whether prestin can be used as a serological biomarker for cochlear damage or stress, we measured prestin quantities in the bloodstreams of mice using ELISA kits from different companies. Wildtype (WT) mice were exposed to different ototoxic treatments, including noise exposure and ototoxic reagents that rapidly kill OHCs. Prestin-knockout (KO) mice were used as a negative control. Our data show that some ELISA kits were not able to detect prestin specifically. The ELISA kit that could detect the prestin protein from cochlear homogenates failed to detect prestin in the bloodstream, despite there being significant damage to OHCs in the cochleae. Furthermore, the optical densities of the serum samples, which correlate to prestin quantities, were significantly influenced by hemolysis in the samples. In conclusion, Prestin from OHCs is not a sensitive and reliable serological biomarker for detecting cochlear damage in mice using ELISA.

Functional Studies of Deafness-Associated Pendrin and Prestin Variants.

Pendrin and prestin are evolutionary-conserved membrane proteins that are essential for normal hearing. Dysfunction of these proteins results in hearing loss in humans, and numerous deafness-associated pendrin and prestin variants have been identified in patients. However, the pathogenic impacts of many of these variants are ambiguous. Here, we report results from our ongoing efforts to experimentally characterize pendrin and prestin variants using in vitro functional assays. With previously established fluorometric anion transport assays, we determined that many of the pendrin variants identified on transmembrane (TM) 10, which contains the essential anion binding site, and on the neighboring TM9 within the core domain resulted in impaired anion transport activity. We also determined the range of functional impairment in three deafness-associated prestin variants by measuring nonlinear capacitance (NLC), a proxy for motor function. Using the results from our functional analyses, we also evaluated the performance of AlphaMissense (AM), a computational tool for predicting the pathogenicity of missense variants. AM prediction scores correlated well with our experimental results; however, some variants were misclassified, underscoring the necessity of experimentally assessing the effects of variants. Together, our experimental efforts provide invaluable information regarding the pathogenicity of deafness-associated pendrin and prestin variants.

In vivo CRISPR-Cas9-mediated DNA chop identifies a cochlear outer hair cell-specific enhancer.

Cochlear outer hair cells (OHCs) are essential for hearing. A short, OHC-specific enhancer is necessary but not yet available for gene therapeutic applications in OHC damage. Such damage is a major cause of deafness. Prestin is a motor protein exclusively expressed in OHCs. We hypothesized that the cis-regulatory DNA fragment deletion of Slc26a5 would affect its expression. We tested this hypothesis by conducting CRISPR/Cas9-mediated large DNA fragment deletion of mouse Slc26a5 intron regions. First, starting from a ~13 kbp fragment, step-by-step, we narrowed down the sequence to a 1.4 kbp segment. By deleting either a 13 kbp or 1.4 kbp fragment, we observed delayed Prestin expression. Second, we showed that 1.4 kbp was an OHC-specific enhancer because enhanced green fluorescent protein (EGFP) was highly and specifically expressed in OHCs in a transgenic mouse where EGFP was driven by the 1.4 kbp segment. More importantly, specific EGFP was also driven by its homologous 398 bp fragment in human Slc26a5. This suggests that the enhancer is likely to be evolutionarily conserved across different species.

The cochlear outer hair cell speed paradox.

Cochlear outer hair cells (OHCs) are among the fastest known biological motors and are essential for high-frequency hearing in mammals. It is commonly hypothesized that OHCs amplify vibrations in the cochlea through cycle-by-cycle changes in length, but recent data suggest OHCs are low-pass filtered and unable to follow high-frequency signals. The fact that OHCs are required for high-frequency hearing but appear to be throttled by slow electromotility is the "OHC speed paradox." The present report resolves this paradox and reveals origins of ultrafast OHC function and power output in the context of the cochlear load. Results demonstrate that the speed of electromotility reflects how fast the cell can extend against the load, and does not reflect the intrinsic speed of the motor element itself or the nearly instantaneous speed at which the coulomb force is transmitted. OHC power output at auditory frequencies is revealed by emergence of an imaginary nonlinear capacitance reflecting the phase of electrical charge displacement required for the motor to overcome the viscous cochlear load.

Evaluation of the relationship between prestin serum biomarker and sensorineural hearing loss: a case-control study.

RESEARCH BACKGROUND AND AIM: There is not any routine serum biomarker for diagnosing hearing loss (HL). An inner ear-specific protein, prestin can be measured as a serum biochemical marker for HL diagnosis. The present study investigates, for the first time, the relationship between prestin serum levels and sensorineural HL (SNHL) in an Iranian population. MATERIALS AND METHODS: In this case-control study, 176 samples were examined in four groups including two control and two SNHL groups of 20-50 and ≥ 50 years with different severities of SNHL. Plasma prestin concentration was measured using Human Prestin (SLC26A5) ELISA Kit. Data analysis was conducted using SPSS v.23 with level of significance as 0.05. RESULTS: Groups with SNHL had higher prestin levels (Mean = 182.29, SD = 71.24) compared to the control groups (Mean = 122.50, SD = 57.1) (P < 0.001). Results of the multinomial logistic regression of relationship between prestin level and SNHL remained significant after controlling intervening variables (P < 0.001 and odds ratio = 1.017 and 95% CI OR: 1.01-1.024). Results of the ordinal logistic regression model revealed that prestin level was significantly associated with the degree of HL (P < 0.001 and Odds ratio = 1.009 and 95% CI and OR: 1.005-1.013), so that the likelihood of HL increased with the rise in prestin levels. The best cutoff point for the 20-50 group was the prestin content of 132.5 pg/ml (sensitivity: 75%, specificity: 70.05%), while for the group of ≥ 50 was as 130 pg/ml (sensitivity: 84.1%, specificity: 68.2%). CONCLUSIONS: Results of the present study revealed that prestin acts as a valuable biomarker for SNHL.

Effect of cisplatin chemotherapy on the inner ear function and serum prestin concentration.

PURPOSE: One of the main side effects of chemotherapy with cisplatin is irreversible sensorineural hearing loss. This study was conducted to assess the correlation between the serum prestin concentration as a potential cochlear biomarker and audiologic findings in patients after cisplatin chemotherapy. METHODS: A total of 52 patients aged 18-75 years were included in this prospective study. All the subjects were recruited from the radiotherapy and oncology center of a tertiary hospital in Rasht, Iran. Audiologic parameters evaluations and serum prestin concentrations were measured at baseline and after 1-3 weeks of chemotherapy. The inner ear function was evaluated by pure-tone audiometry (PTA) and distortion product of otoacoustic emission (DPOAE). A repeated-measure analysis of variance was performed to evaluate the relationship between the PTA, DPOAE, serum prestin concentration and cumulative cisplatin dose. RESULTS: Fifty-two patients (36 females) participated in this study. The PTA results showed that ototoxicity was more frequent among the patients with a high cumulative dose of cisplatin (χ2 trend = 15.25; P < 0.001). DPOAE responses revealed that 38.5% of the patients had ototoxicity change after 40-80 mg of cisplatin administration. After receiving 40-80 mg of cisplatin, serum prestin concentration increased from 130 to 230 pg/ml. There is a significant positive correlation between prestin concentration after receiving more than 80 mg of cisplatin and the ototoxic changes in the DPOAE response. CONCLUSION: The present study showed correlations between prestin concentrations and ototoxicity diagnosis by DPOAE findings. An early rise in prestin concentration is particularly important and an early sign of hearing loss. Future studies are recommended to investigate the effect of varying doses of cisplatin on prestin concentration and any association between ototoxicity and clinicopathological features.

Identification and characterization of amphibian SLC26A5 using RNA-Seq.

BACKGROUND: Prestin (SLC26A5) is responsible for acute sensitivity and frequency selectivity in the vertebrate auditory system. Limited knowledge of prestin is from experiments using site-directed mutagenesis or domain-swapping techniques after the amino acid residues were identified by comparing the sequence of prestin to those of its paralogs and orthologs. Frog prestin is the only representative in amphibian lineage and the studies of it were quite rare with only one species identified. RESULTS: Here we report a new coding sequence of SLC26A5 for a frog species, Rana catesbeiana (the American bullfrog). In our study, the SLC26A5 gene of Rana has been mapped, sequenced and cloned successively using RNA-Seq. We measured the nonlinear capacitance (NLC) of prestin both in the hair cells of Rana's inner ear and HEK293T cells transfected with this new coding gene. HEK293T cells expressing Rana prestin showed electrophysiological features similar to that of hair cells from its inner ear. Comparative studies of zebrafish, chick, Rana and an ancient frog species showed that chick and zebrafish prestin lacked NLC. Ancient frog's prestin was functionally different from Rana. CONCLUSIONS: We mapped and sequenced the SLC26A5 of the Rana catesbeiana from its inner ear cDNA using RNA-Seq. The Rana SLC26A5 cDNA was 2292 bp long, encoding a polypeptide of 763 amino acid residues, with 40% identity to mammals. This new coding gene could encode a functionally active protein conferring NLC to both frog HCs and the mammalian cell line. While comparing to its orthologs, the amphibian prestin has been evolutionarily changing its function and becomes more advanced than avian and teleost prestin.

Simultaneous zygotic inactivation of multiple genes in mouse through CRISPR/Cas9-mediated base editing.

In vivo genetic mutation has become a powerful tool for dissecting gene function; however, multi-gene interaction and the compensatory mechanisms involved can make findings from single mutations, at best difficult to interpret, and, at worst, misleading. Hence, it is necessary to establish an efficient way to disrupt multiple genes simultaneously. CRISPR/Cas9-mediated base editing disrupts gene function by converting a protein-coding sequence into a stop codon; this is referred to as CRISPR-stop. Its application in generating zygotic mutations has not been well explored yet. Here, we first performed a proof-of-principle test by disrupting Atoh1, a gene crucial for auditory hair cell generation. Next, we individually mutated vGlut3 (Slc17a8), otoferlin (Otof) and prestin (Slc26a5), three genes needed for normal hearing function. Finally, we successfully disrupted vGlut3, Otof and prestin simultaneously. Our results show that CRISPR-stop can efficiently generate single or triple homozygous F0 mouse mutants, bypassing laborious mouse breeding. We believe that CRISPR-stop is a powerful method that will pave the way for high-throughput screening of mouse developmental and functional genes, matching the efficiency of methods available for model organisms such as Drosophila.

HCN channels in the mammalian cochlea: Expression pattern, subcellular location, and age-dependent changes.

Neuronal diversity in the cochlea is largely determined by ion channels. Among voltage-gated channels, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels open with hyperpolarization and depolarize the cell until the resting membrane potential. The functions for hearing are not well elucidated and knowledge about localization is controversial. We created a detailed map of subcellular location and co-expression of all four HCN subunits across different mammalian species including CBA/J, C57Bl/6N, Ly5.1 mice, guinea pigs, cats, and human subjects. We correlated age-related hearing deterioration in CBA/J and C57Bl/6N with expression levels of HCN1, -2, and -4 in individual auditory neurons from the same cohort. Spatiotemporal expression during murine postnatal development exposed HCN2 and HCN4 involvement in a critical phase of hair cell innervation. The huge diversity of subunit composition, but lack of relevant heteromeric pairing along the perisomatic membrane and axon initial segments, highlighted an active role for auditory neurons. Neuron clusters were found to be the hot spots of HCN1, -2, and -4 immunostaining. HCN channels were also located in afferent and efferent fibers of the sensory epithelium. Age-related changes on HCN subtype expression were not uniform among mice and could not be directly correlated with audiometric data. The oldest mice groups revealed HCN channel up- or downregulation, depending on the mouse strain. The unexpected involvement of HCN channels in outer hair cell function where HCN3 overlaps prestin location emphasized the importance for auditory function. A better understanding may open up new possibilities to tune neuronal responses evoked through electrical stimulation by cochlear implants.

Prestin and otolin-1 proteins in the hearing loss of adults chronically exposed to lead.

BACKGROUND: Some studies in animal models and humans suggest that exposure to lead is associated with hearing loss. Lead can reach the inner ear through the blood circulation; evidence suggests that lead could accumulate in the inner ear, causing inner ear damage. AIM: To evaluate prestin and otolin-1 protein levels and their relationship with an increased hearing threshold in participants exposed to lead. METHODS: We conducted a cross-sectional study with 315 participants from Tlaxcala, Mexico. Blood lead levels (BPb) were evaluated by graphite furnace atomic absorption spectrometry. Serum prestin and otolin-1 were quantified using ELISA. Auditory function at frequencies of 0.125 to 8 kHz was evaluated in a soundproof chamber. RESULTS: Participants were classified according to BPb: group I (<10 µg/dL) had a median BPb of 6 µg/dL and prestin levels of 11.06 ng/mL. While participants in group II (≥10 µg/dL) had a median of BPb 20.7 µg/dL (p < 0.05) and prestin levels of 0.15 ng/mL (p < 0.001). Participants in both groups showed a normal hearing. Otolin-1 levels were higher for participants with normal hearing and lower for participants with hearing loss in both groups, p > 0.05. Multiple linear regression models predict an average decrease of 0.17 to 0.26 ng/mL in prestin levels per decibel increase for the frequencies evaluated. CONCLUSIONS: Participants with high BPb showed an increase in hearing threshold, and prestin levels decreased proportionally to the hearing threshold increase. This is the first study to evaluate prestin as a potential biomarker for hearing damage, evaluated by audiometry, in participants with lead exposure.

Comparative Molecular Dynamics Investigation of the Electromotile Hearing Protein Prestin.

The mammalian protein prestin is expressed in the lateral membrane wall of the cochlear hair outer cells and is responsible for the electromotile response of the basolateral membrane, following hyperpolarisation or depolarisation of the cells. Its impairment marks the onset of severe diseases, like non-syndromic deafness. Several studies have pointed out possible key roles of residues located in the Transmembrane Domain (TMD) that differentiate mammalian prestins as incomplete transporters from the other proteins belonging to the same solute-carrier (SLC) superfamily, which are classified as complete transporters. Here, we exploit the homology of a prototypical incomplete transporter (rat prestin, rPres) and a complete transporter (zebrafish prestin, zPres) with target structures in the outward open and inward open conformations. The resulting models are then embedded in a model membrane and investigated via a rigorous molecular dynamics simulation protocol. The resulting trajectories are analyzed to obtain quantitative descriptors of the equilibration phase and to assess a structural comparison between proteins in different states, and between different proteins in the same state. Our study clearly identifies a network of key residues at the interface between the gate and the core domains of prestin that might be responsible for the conformational change observed in complete transporters and hindered in incomplete transporters. In addition, we study the pathway of Cl- ions in the presence of an applied electric field towards their putative binding site in the gate domain. Based on our simulations, we propose a tilt and shift mechanism of the helices surrounding the ion binding cavity as the working principle of the reported conformational changes in complete transporters.

Inner Ear Genes Underwent Positive Selection and Adaptation in the Mammalian Lineage.

The mammalian inner ear possesses functional and morphological innovations that contribute to its unique hearing capacities. The genetic bases underlying the evolution of this mammalian landmark are poorly understood. We propose that the emergence of morphological and functional innovations in the mammalian inner ear could have been driven by adaptive molecular evolution. In this work, we performed a meta-analysis of available inner ear gene expression data sets in order to identify genes that show signatures of adaptive evolution in the mammalian lineage. We analyzed ∼1,300 inner ear expressed genes and found that 13% show signatures of positive selection in the mammalian lineage. Several of these genes are known to play an important function in the inner ear. In addition, we identified that a significant proportion of genes showing signatures of adaptive evolution in mammals have not been previously reported to participate in inner ear development and/or physiology. We focused our analysis in two of these genes: STRIP2 and ABLIM2 by generating null mutant mice and analyzed their auditory function. We found that mice lacking Strip2 displayed a decrease in neural response amplitudes. In addition, we observed a reduction in the number of afferent synapses, suggesting a potential cochlear neuropathy. Thus, this study shows the usefulness of pursuing a high-throughput evolutionary approach followed by functional studies to track down genes that are important for inner ear function. Moreover, this approach sheds light on the genetic bases underlying the evolution of the mammalian inner ear.

Effects of imidacloprid, a neonicotinoid insecticide, on the echolocation system of insectivorous bats.

Imidacloprid, a widely used neonicotinoid insecticide, has led to a decline in the honey bee population worldwide. An invertebrate insect prey with neonicotinoid toxicity can adversely affect insectivores, such as echolocating bats. The aim of the current study was to examined whether imidacloprid toxicity may interfere echolocation system such as vocal, auditory, orientation, and spatial memory systems in the insectivorous bat. By comparing the ultrasound spectrum, auditory brainstem-evoked potential, and flight trajectory, we found that imidacloprid toxicity may interfere functions in vocal, auditory, orientation, and spatial memory system of insectivorous bats (Hipposideros armiger terasensis). As suggested from immunohistochemistry and western blots evidences, we found that insectivorous bats after suffering imidacloprid toxicity may decrease vocal-related FOXP2 expressions in the superior colliculus, auditory-related prestin expressions in the cochlea, and the auditory-related otoferlin expressions in the cochlea and the inferior colliculus, and cause inflammation and mitochondrial dysfunction-related apoptosis in the hippocampal CA1 and medial entorhinal cortex. These results may provide a reasonable explanation about imidacloprid-induced interference of echolocation system in insectivorous bats.