Anping Xia

Wnt signaling induces proliferation of sensory precursors in the postnatal mouse cochlea

Inner ear hair cells are specialized sensory cells essential for auditory function. Previous studies have shown that the sensory epithelium is postmitotic, but it harbors cells that can behave as progenitor cells in vitro, including the ability to form new hair cells. Lgr5, a Wnt target gene, marks distinct supporting cell types in the neonatal cochlea. Here, we tested the hypothesis that Lgr5+ cells are Wnt-responsive sensory precursor cells. In contrast to their quiescent in vivo behavior, Lgr5+ cells isolated by flow cytometry from neonatal Lgr5EGFP-CreERT2/+ mice proliferated and formed clonal colonies. After 10 d in culture, new sensory cells formed and displayed specific hair cell markers (myo7a, calretinin, parvalbumin, myo6) and stereocilia-like structures expressing F-actin and espin. In comparison with other supporting cells, Lgr5+ cells were enriched precursors to myo7a+ cells, most of which formed without mitotic division. Treatment with Wnt agonists increased proliferation and colony-formation capacity. Conversely, small-molecule inhibitors of Wnt signaling suppressed proliferation without compromising the myo7a+ cells formed by direct differentiation. In vivo lineage tracing supported the idea that Lgr5+ cells give rise to myo7a+ hair cells in the neonatal Lgr5EGFP-CreERT2/+ cochlea. In addition, overexpression of β-catenin initiated proliferation and led to transient expansion of Lgr5+ cells within the cochlear sensory epithelium. These results suggest that Lgr5 marks sensory precursors and that Wnt signaling can promote their proliferation and provide mechanistic insights into Wnt-responsive progenitor cells during sensory organ development.

Tuning of the outer hair cell motor by membrane cholesterol

Cholesterol affects diverse biological processes, in many cases by modulating the function of integral membrane proteins. We observed that alterations of cochlear cholesterol modulate hearing in mice. Mammalian hearing is powered by outer hair cell (OHC) electromotility, a membrane-based motor mechanism that resides in the OHC lateral wall. We show that membrane cholesterol decreases during maturation of OHCs. To study the effects of cholesterol on hearing at the molecular level, we altered cholesterol levels in the OHC wall, which contains the membrane protein prestin. We show a dynamic and reversible relationship between membrane cholesterol levels and voltage dependence of prestin-associated charge movement in both OHCs and prestin-transfected HEK 293 cells. Cholesterol levels also modulate the distribution of prestin within plasma membrane microdomains and affect prestin self-association in HEK 293 cells. These findings indicate that alterations in membrane cholesterol affect prestin function and functionally tune the outer hair cell.

Atoh1-lineal neurons are required for hearing and for the survival of neurons in the spiral ganglion and brainstem accessory auditory nuclei

Atoh1 is a basic helix–loop–helix transcription factor necessary for the specification of inner ear hair cells and central auditory system neurons derived from the rhombic lip. We used the Cre–loxP system and two Cre-driver lines (Egr2Cre and Hoxb1Cre ) to delete Atoh1 from different regions of the cochlear nucleus (CN) and accessory auditory nuclei (AAN). Adult Atoh1-conditional knock-out mice (Atoh1CKO ) are behaviorally deaf, have diminished auditory brainstem evoked responses, and have disrupted CN and AAN morphology and connectivity. In addition, Egr2; Atoh1CKO mice lose spiral ganglion neurons in the cochlea and AAN neurons during the first 3 d of life, revealing a novel critical period in the development of these neurons. These new mouse models of predominantly central deafness illuminate the importance of the CN for support of a subset of peripheral and central auditory neurons.

Deficient forward transduction and enhanced reverse transduction in the alpha tectorin C1509G human hearing loss mutation

SUMMARY Most forms of hearing loss are associated with loss of cochlear outer hair cells (OHCs). OHCs require the tectorial membrane (TM) for stereociliary bundle stimulation (forward transduction) and active feedback (reverse transduction). Alpha tectorin is a protein constituent of the TM and the C1509G mutation in alpha tectorin in humans results in autosomal dominant hearing loss. We engineered and validated this mutation in mice and found that the TM was shortened in heterozygous TectaC1509G/+ mice, reaching only the first row of OHCs. Thus, deficient forward transduction renders OHCs within the second and third rows non-functional, producing partial hearing loss. Surprisingly, both TectaC1509G/+ and TectaC1509G/C1509G mice were found to have increased reverse transduction as assessed by sound- and electrically-evoked otoacoustic emissions. We show that an increase in prestin, a protein necessary for electromotility, in all three rows of OHCs underlies this phenomenon. This mouse model demonstrates a human hearing loss mutation in which OHC function is altered through a non-cell-autonomous variation in prestin.

Mechanisms of hearing loss after blast injury to the ear.

Given the frequent use of improvised explosive devices (IEDs) around the world, the study of traumatic blast injuries is of increasing interest. The ear is the most common organ affected by blast injury because it is the body’s most sensitive pressure transducer. We fabricated a blast chamber to re-create blast profiles similar to that of IEDs and used it to develop a reproducible mouse model to study blast-induced hearing loss. The tympanic membrane was perforated in all mice after blast exposure and found to heal spontaneously. Micro-computed tomography demonstrated no evidence for middle ear or otic capsule injuries; however, the healed tympanic membrane was thickened. Auditory brainstem response and distortion product otoacoustic emission threshold shifts were found to be correlated with blast intensity. As well, these threshold shifts were larger than those found in control mice that underwent surgical perforation of their tympanic membranes, indicating cochlear trauma. Histological studies one week and three months after the blast demonstrated no disruption or damage to the intra-cochlear membranes. However, there was loss of outer hair cells (OHCs) within the basal turn of the cochlea and decreased spiral ganglion neurons (SGNs) and afferent nerve synapses. Using our mouse model that recapitulates human IED exposure, our results identify that the mechanisms underlying blast-induced hearing loss does not include gross membranous rupture as is commonly believed. Instead, there is both OHC and SGN loss that produce auditory dysfunction.

Tympanic border cells are Wnt-responsive and can act as progenitors for postnatal mouse cochlear cells

Permanent hearing loss is caused by the irreversible damage of cochlear sensory hair cells and nonsensory supporting cells. In the postnatal cochlea, the sensory epithelium is terminally differentiated, whereas tympanic border cells (TBCs) beneath the sensory epithelium are proliferative. The functions of TBCs are poorly characterized. Using an Axin2lacZ Wnt reporter mouse, we found transient but robust Wnt signaling and proliferation in TBCs during the first 3 postnatal weeks, when the number of TBCs decreases. In vivo lineage tracing shows that a subset of hair cells and supporting cells is derived postnatally from Axin2-expressing TBCs. In cochlear explants, Wnt agonists stimulated the proliferation of TBCs, whereas Wnt inhibitors suppressed it. In addition, purified Axin2lacZ cells were clonogenic and self-renewing in culture in a Wnt-dependent manner, and were able to differentiate into hair cell-like and supporting cell-like cells. Taken together, our data indicate that Axin2-positive TBCs are Wnt responsive and can act as precursors to sensory epithelial cells in the postnatal cochlea.

Quantitative imaging of cochlear soft tissues in wild-type and hearing-impaired transgenic mice by spectral domain optical coherence tomography

Human hearing loss often occurs as a result of damage or malformations to the functional soft tissues within the cochlea, but these changes are not appreciable with current medical imaging modalities. We sought to determine whether optical coherence tomography (OCT) could assess the soft tissue structures relevant to hearing using mouse models. We imaged excised cochleae with an altered tectorial membrane and during normal development. The soft tissue structures and expected anatomical variations were visible using OCT, and quantitative measurements confirmed the ability to detect critical changes relevant to hearing.

In vivo vibrometry inside the apex of the mouse cochlea using spectral domain optical coherence tomography

Sound transduction within the auditory portion of the inner ear, the cochlea, is a complex nonlinear process. The study of cochlear mechanics in large rodents has provided important insights into cochlear function. However, technological and experimental limitations have restricted studies in mice due to their smaller cochlea. These challenges are important to overcome because of the wide variety of transgenic mouse strains with hearing loss mutations that are available for study. To accomplish this goal, we used spectral domain optical coherence tomography to visualize and measure sound-induced vibrations of intracochlear tissues. We present, to our knowledge, the first vibration measurements from the apex of an unopened mouse cochlea.

Two-Dimensional Cochlear Micromechanics Measured In Vivo Demonstrate Radial Tuning within the Mouse Organ of Corti

The exquisite sensitivity and frequency discrimination of mammalian hearing underlie the ability to understand complex speech in noise. This requires force generation by cochlear outer hair cells (OHCs) to amplify the basilar membrane traveling wave; however, it is unclear how amplification is achieved with sharp frequency tuning. Here we investigated the origin of tuning by measuring sound-induced 2-D vibrations within the mouse organ of Corti in vivo. Our goal was to determine the transfer function relating the radial shear between the structures that deflect the OHC bundle, the tectorial membrane and reticular lamina, to the transverse motion of the basilar membrane. We found that, after normalizing their responses to the vibration of the basilar membrane, the radial vibrations of the tectorial membrane and reticular lamina were tuned. The radial tuning peaked at a higher frequency than transverse basilar membrane tuning in the passive, postmortem condition. The radial tuning was similar in dead mice, indicating that this reflected passive, not active, mechanics. These findings were exaggerated in TectaC1509G/C1509G mice, where the tectorial membrane is detached from OHC stereocilia, arguing that the tuning of radial vibrations within the hair cell epithelium is distinct from tectorial membrane tuning. Together, these results reveal a passive, frequency-dependent contribution to cochlear filtering that is independent of basilar membrane filtering. These data argue that passive mechanics within the organ of Corti sharpen frequency selectivity by defining which OHCs enhance the vibration of the basilar membrane, thereby tuning the gain of cochlear amplification. SIGNIFICANCE STATEMENT Outer hair cells amplify the traveling wave within the mammalian cochlea. The resultant gain and frequency sharpening are necessary for speech discrimination, particularly in the presence of background noise. Here we measured the 2-D motion of the organ of Corti in mice and found that the structures that stimulate the outer hair cell stereocilia, the tectorial membrane and reticular lamina, were sharply tuned in the radial direction. Radial tuning was similar in dead mice and in mice lacking a tectorial membrane. This suggests that radial tuning comes from passive mechanics within the hair cell epithelium, and that these mechanics, at least in part, may tune the gain of cochlear amplification.

Prestin Regulation and Function in Residual Outer Hair Cells after Noise-Induced Hearing Loss

The outer hair cell (OHC) motor protein prestin is necessary for electromotility, which drives cochlear amplification and produces exquisitely sharp frequency tuning. TectaC1509G transgenic mice have hearing loss, and surprisingly have increased OHC prestin levels. We hypothesized, therefore, that prestin up-regulation may represent a generalized response to compensate for a state of hearing loss. In the present study, we sought to determine the effects of noise-induced hearing loss on prestin expression. After noise exposure, we performed cytocochleograms and observed OHC loss only in the basal region of the cochlea. Next, we patch clamped OHCs from the apical turn (9–12 kHz region), where no OHCs were lost, in noise-exposed and age-matched control mice. The non-linear capacitance was significantly higher in noise-exposed mice, consistent with higher functional prestin levels. We then measured prestin protein and mRNA levels in whole-cochlea specimens. Both Western blot and qPCR studies demonstrated increased prestin expression after noise exposure. Finally, we examined the effect of the prestin increase in vivo following noise damage. Immediately after noise exposure, ABR and DPOAE thresholds were elevated by 30–40 dB. While most of the temporary threshold shifts recovered within 3 days, there were additional improvements over the next month. However, DPOAE magnitudes, basilar membrane vibration, and CAP tuning curve measurements from the 9–12 kHz cochlear region demonstrated no differences between noise-exposed mice and control mice. Taken together, these data indicate that prestin is up-regulated by 32–58% in residual OHCs after noise exposure and that the prestin is functional. These findings are consistent with the notion that prestin increases in an attempt to partially compensate for reduced force production because of missing OHCs. However, in regions where there is no OHC loss, the cochlea is able to compensate for the excess prestin in order to maintain stable auditory thresholds and frequency discrimination.