Chromatin remodeler CHD7 is critical for cochlear morphogenesis and neurosensory patterning.

Epigenetic regulation of gene transcription by chromatin remodeling proteins has recently emerged as an important contributing factor in inner ear development. Pathogenic variants in CHD7, the gene encoding Chromodomain Helicase DNA binding protein 7, cause CHARGE syndrome, which presents with malformations in the developing ear. Chd7 is broadly expressed in the developing mouse otocyst and mature auditory epithelium, yet the pathogenic effects of Chd7 loss in the cochlea are not well understood. Here we characterized cochlear epithelial phenotypes in mice with deletion of Chd7 throughout the otocyst (using Foxg1Cre/+ and Pax2Cre), in the otic mesenchyme (using TCre), in hair cells (using Atoh1Cre), in developing neuroblasts (using NgnCre), or in spiral ganglion neurons (using ShhCre/+). Pan-otic deletion of Chd7 resulted in shortened cochleae with aberrant projections and axonal looping, disorganized, supernumerary hair cells at the apical turn and a narrowed epithelium with missing hair cells in the middle region. Deletion of Chd7 in the otic mesenchyme had no effect on overall cochlear morphology. Loss of Chd7 in hair cells did not disrupt their formation or organization of the auditory epithelium. Similarly, absence of Chd7 in spiral ganglion neurons had no effect on axonal projections. In contrast, deletion of Chd7 in developing neuroblasts led to smaller spiral ganglia and disorganized cochlear neurites. Together, these observations reveal dosage-, tissue-, and time-sensitive cell autonomous roles for Chd7 in cochlear elongation and cochlear neuron organization, with minimal functions for Chd7 in hair cells. These studies provide novel information about roles for Chd7 in development of auditory neurons.

microRNA-224 regulates Pentraxin 3, a component of the humoral arm of innate immunity, in inner ear inflammation.

microRNAs (miRNAs) are regulators of differentiation and development of inner ear cells. Mutations in miRNAs lead to deafness in humans and mice. Among inner ear pathologies, inflammation may lead to structural and neuronal defects and eventually to hearing loss and vestibular dysfunction. While the genetic factors of these pathways have not been defined, autoimmunity participates in these processes. We report that inflammatory stimuli in the inner ear induce activation of the innate immune system via miR-224 and pentraxin 3 (Ptx3). miR-224 is a transcriptional target of nuclear factor κB, a key mediator of innate immunity. Ptx3 is a regulator of the immune response. It is released in response to inflammation and regulated by nuclear factor κB. We show that miR-224 and Ptx3 are expressed in the inner ear and we demonstrate that miR-224 targets Ptx3. As a model of the innate immune response, we injected lipopolysaccharide into the scala tympani of mouse inner ears. This resulted in changes in the levels of miR-224 and Ptx3, in addition to activation of the complement system, as measured by immune cell infiltration and activated C3. This suggests that while miR-224 regulates Ptx3 under normal conditions, upon inflammation, both are recruited to offer a front line of defense in acting as responders to inflammation in the inner ear. miR-224 diminishes the innate immune response by down-regulating Ptx3 expression, while Ptx3 stimulates the innate immune response. An understanding of the molecular components of the inflammatory pathway may help develop therapeutics for reducing inflammation associated with inner ear injury.

A mechanistic pharmacokinetic/pharmacodynamic model of factor D inhibition in cynomolgus monkeys by lampalizumab for the treatment of geographic atrophy.

Lampalizumab is an antigen-binding fragment of a humanized monoclonal antibody against complement factor D (CFD), a rate-limiting enzyme in the activation and amplification of the alternative complement pathway (ACP), which is in phase III clinical trials for the treatment of geographic atrophy. Understanding of the pharmacokinetics, pharmacodynamics, and biodistribution of lampalizumab following intravitreal administration in the ocular compartments and systemic circulation is limited but crucial for selecting doses that provide optimal efficacy and safety. Here, we sought to construct a semimechanistic and integrated ocular-systemic pharmacokinetic-pharmacodynamic model of lampalizumab in the cynomolgus monkey to provide a quantitative understanding of the ocular and systemic disposition of lampalizumab and CFD inhibition. The model takes into account target-mediated drug disposition, target turnover, and drug distribution across ocular tissues and systemic circulation. Following intravitreal administration, lampalizumab achieves rapid equilibration across ocular tissues. Lampalizumab ocular elimination is relatively slow, with a τ1/2 of approximately 3 days, whereas systemic elimination is rapid, with a τ1/2 of 0.8 hours. Target-independent linear clearance is predominant in the eye, whereas target-mediated clearance is predominant in the systemic circulation. Systemic CFD synthesis was estimated to be high (7.8 mg/day); however, the amount of CFD entering the eye due to influx from the systemic circulation was small (<10%) compared with the lampalizumab dose and is thus expected to have an insignificant impact on the clinical dose-regimen decision. Our findings support the clinical use of intravitreal lampalizumab to achieve significant ocular ACP inhibition while maintaining low systemic exposure and minimal systemic ACP inhibition.

Conditioning the cochlea to facilitate survival and integration of exogenous cells into the auditory epithelium.

The mammalian auditory epithelium (AE) cannot replace supporting cells and hair cells once they are lost. Therefore, sensorineural hearing loss associated with missing cells is permanent. This inability to regenerate critical cell types makes the AE a potential target for cell replacement therapies such as stem cell transplantation. Inserting stem cells into the AE of deaf ears is a complicated task due to the hostile, high potassium environment of the scala media in the cochlea, and the robust junctional complexes between cells in the AE that resist stem cell integration. Here, we evaluate whether temporarily reducing potassium levels in the scala media and disrupting the junctions in the AE make the cochlear environment more receptive and facilitate survival and integration of transplanted cells. We used sodium caprate to transiently disrupt the AE junctions, replaced endolymph with perilymph, and blocked stria vascularis pumps with furosemide. We determined that these three steps facilitated survival of HeLa cells in the scala media for at least 7 days and that some of the implanted cells formed a junctional contact with native AE cells. The data suggest that manipulation of the cochlear environment facilitates survival and integration of exogenously transplanted HeLa cells in the scala media.

Complement inhibition in cynomolgus monkeys by anti-factor d antigen-binding fragment for the treatment of an advanced form of dry age-related macular degeneration.

Anti-factor D (AFD; FCFD4514S, lampalizumab) is a humanized IgG Fab fragment directed against factor D (fD), a rate-limiting serine protease in the alternative complement pathway (AP). Evaluation of AFD as a potential intravitreal (IVT) therapeutic for dry age-related macular degeneration patients with geographic atrophy (GA) is ongoing. However, it is unclear whether IVT administration of AFD can affect systemic AP activation and potentially compromise host-immune responses. We characterized the pharmacologic properties of AFD and assessed the effects of AFD administered IVT (2 or 20 mg) or intravenous (0.2, 2, or 20 mg) on systemic complement activity in cynomolgus monkeys. For the IVT groups, serum AP activity was reduced for the 20 mg dose group between 2 and 6 hours postinjection. For the intravenous groups, AFD inhibited systemic AP activity for periods of time ranging from 5 minutes (0.2 mg group) to 3 hours (20 mg group). Interestingly, the concentrations of total serum fD increased up to 10-fold relative to predose levels following administration of AFD. Furthermore, AFD was found to inhibit systemic AP activity only when the molar concentration of AFD exceeded that of fD. This occurred in cynomolgus monkeys at serum AFD levels ≥2 µg/ml, a concentration 8-fold greater than the maximum serum concentration observed following a single 10 mg IVT dose in a clinical investigation in patients with GA. Based on these findings, the low levels of serum AFD resulting from IVT administration of a clinically relevant dose are not expected to appreciably affect systemic AP activity.

Combinatorial Atoh1, Gfi1, Pou4f3, and Six1 gene transfer induces hair cell regeneration in the flat epithelium of mature guinea pigs.

Flat epithelium (FE) is a condition characterized by the loss of both hair cells (HCs) and supporting cells and the transformation of the organ of Corti into a simple flat or cuboidal epithelium, which can occur after severe cochlear insults. The transcription factors Gfi1, Atoh1, Pou4f3, and Six1 (GAPS) play key roles in HC differentiation and survival in normal ears. Previous work using a single transcription factor, Atoh1, to induce HC regeneration in mature ears in vivo usually produced very few cells and failed to produce HCs in severely damaged organs of Corti, especially those with FE. Studies in vitro suggested combinations of transcription factors may be more effective than any single factor, thus the current study aims to examine the effect of co-overexpressing GAPS genes in deafened mature guinea pig cochleae with FE. Deafening was achieved through the infusion of neomycin into the perilymph, leading to the formation of FE and substantial degeneration of nerve fibers. Seven days post neomycin treatment, adenovirus vectors carrying GAPS were injected into the scala media and successfully expressed in the FE. One or two months following GAPS inoculation, cells expressing Myosin VIIa were observed in regions under the FE (located at the scala tympani side of the basilar membrane), rather than within the FE. The number of cells, which we define as induced HCs (iHCs), was not significantly different between one and two months, but the larger N at two months made it more apparent that there were significantly more iHCs in GAPS treated animals than in controls. Additionally, qualitative observations indicated that ears with GAPS gene expression in the FE had more nerve fibers than FE without the treatment. In summary, our results showed that co-overexpression of GAPS enhances the potential for HC regeneration in a severe lesion model of FE.

Role of auditory feedback for vocal production learning in the Egyptian fruit bat.

Some species have evolved the ability to use the sense of hearing to modify existing vocalizations, or even create new ones, which enlarges their repertoires and results in complex communication systems.1 This ability corresponds to various forms of vocal production learning that are all possessed by humans and independently displayed by distantly related vertebrates.1,2,3,4,5,6,7 Among mammals, a few species, including the Egyptian fruit bat,8,9,10 would possess such vocal production learning abilities.7 Yet the necessity of an intact auditory system for the development of the Egyptian fruit bat typical vocal repertoire has not been tested. Furthermore, a systematic causal examination of learned and innate aspects of the entire repertoire has never been performed in any vocal learner. Here we addressed these gaps by eliminating pups' sense of hearing at birth and assessing its effects on vocal production in adulthood. The deafening treatment enabled us to both causally test these bats' vocal learning ability and discern learned from innate aspects of their vocalizations. Leveraging wireless individual audio recordings from freely interacting adults, we show that a subset of the Egyptian fruit bat vocal repertoire necessitates auditory feedback. Intriguingly, these affected vocalizations belong to different acoustic groups in the vocal repertoire of males and females. These findings open the possibilities for targeted studies of the mammalian neural circuits that enable sexually dimorphic forms of vocal learning.

Role of auditory feedback for vocal production learning in the Egyptian fruit-bat.

Some species have evolved the ability to use the sense of hearing to modify existing vocalizations, or even create new ones. This ability corresponds to various forms of vocal production learning that are all possessed by humans, and independently displayed by distantly related vertebrates. Among mammals, a few species, including the Egyptian fruit-bat, would possess such vocal production learning abilities. Yet the necessity of an intact auditory system for the development of the Egyptian fruit-bat typical vocal repertoire has not been tested. Furthermore, a systematic causal examination of learned and innate aspects of the entire repertoire has never been performed in any vocal learner. Here we addressed these gaps by eliminating pups' sense of hearing at birth and assessing its effects on vocal production in adulthood. The deafening treatment enabled us to both causally test these bats vocal learning ability and discern learned from innate aspects of their vocalizations. Leveraging wireless individual audio recordings from freely interacting adults, we show that a subset of the Egyptian fruit-bat vocal repertoire necessitates auditory feedback. Intriguingly, these affected vocalizations belong to different acoustic groups in the vocal repertoire of males and females. These findings open the possibilities for targeted studies of the mammalian neural circuits that enable sexually dimorphic forms of vocal learning.

Anti-neuropilin-1 (MNRP1685A): unexpected pharmacokinetic differences across species, from preclinical models to humans.

PURPOSE: To compare the pharmacokinetics (PK) of MNRP1685A, a human monoclonal antibody (mAb) against neuropilin-1 (NRP1), in mice, rats, monkeys, and cancer patients from a Phase I study to model with parallel linear and nonlinear clearances. METHODS: Binding characteristics of MNRP1685A in different species were evaluated using surface plasmon resonance technology. PK profiles of MNRP1685A after single and/or multiple doses in different species were analyzed using population analysis. PK parameters were compared across species. RESULTS: MNRP1685A binds to NRP1 in all four species tested. Consistent with the wide expression of NRP1, MNRP1685A demonstrated pronounced non-linear PK over a wide dose range. PK profiles are best described by a two-compartment model with parallel linear and nonlinear clearances. Model-derived PK parameters suggest similar in-vivo target expression levels and binding affinity to target across all species tested. However, compared to typical human/humanized mAbs, non-specific clearance of MNRP1685A was faster in mice, rats, and humans (60.3, 19.4, and 8.5 ml/day/kg), but not in monkeys (3.22 ml/day/kg). CONCLUSIONS: Monkey PK properly predicted the target-mediated clearance of MNRP1685A but underestimated its non-specific clearance in humans. This unique PK property warrants further investigation of underlying mechanisms.

Population pharmacokinetic and pharmacodynamic modeling of MNRP1685A in cynomolgus monkeys using two-target quasi-steady-state (QSS) model.

MNRP1685A (anti-NRP1) is a fully human IgG1 monoclonal antibody against neuropilin-1 (NRP1), a protein necessary for blood vessel maturation. MNRP1685A binds to free membrane-bound NRP1 (mNRP1) and circulating NRP1 (cNRP1). Total cNRP1 increased in a dose-dependent manner following anti-NRP1 administration in mice, rats, and monkeys. The purpose of this study is to develop a mechanism-based model to simultaneously describe the kinetics of both unbound drug (MNRP1685A) and total cNRP1 in cynomolgus monkeys. Pharmacokinetic (PK) and pharmacodynamic (PD) profiles after single- or multiple-dose administrations were well described by the two-target quasi-steady-state (QSS) model. The estimated nonspecific clearance was 3.26 mL/day/kg and central compartment volume was 38.2 mL/kg. The maximum elimination rate for mNRP1-mediated disposition was 98.8 nM/day. The synthesis rate (3.8 nM/day), degradation rate constant (1.53 day(-1)), and complex elimination rate constant (0.260 day(-1)) for cNRP1 were also derived from the model. QSS constants were 6.94 nM for mNRP1 and 2.8 nM for cNRP1. The results suggest that cNRP1 has minimal effect on MNRP1685A PK while mNRP1 plays a major role in the target-mediated drug disposition. This finding is favorable as the desired pharmacological target is mNRP1, rather than cNRP1. The two-target QSS model provides mechanistic understanding of the nonlinear PK of MNRP1685A. Based on the model prediction, the free drug concentrations to maintain free mNRP1 and cNRP1 below 10% of baseline level are 10 and 20 µg/mL, respectively. This serves as a target concentration for clinical dose selection, assuming cynomolgus monkeys are predictive for humans.

Loss of the chromatin remodeler CHD7 impacts glial cells and myelination in the mouse cochlear spiral ganglion.

CHARGE syndrome is a multiple anomaly developmental disorder characterized by a variety of sensory deficits, including sensorineural hearing loss of unknown etiology. Most cases of CHARGE are caused by heterozygous pathogenic variants in CHD7, the gene encoding Chromodomain DNA-binding Protein 7 (CHD7), a chromatin remodeler important for the development of neurons and glial cells. Previous studies in the Chd7Gt/+ mouse model of CHARGE syndrome showed substantial neuron loss in the early stages of the developing inner ear that are compensated for by mid-gestation. In this study, we sought to determine if early developmental delays caused by Chd7 haploinsufficiency affect neurons, glial cells, and inner hair cell innervation in the mature cochlea. Analysis of auditory brainstem response recordings in Chd7Gt/+ adult animals showed elevated thresholds at 4 kHz and 16 kHz, but no differences in ABR Wave I peak latency or amplitude compared to wild type controls. Proportions of neurons in the Chd7Gt/+ adult spiral ganglion and densities of nerve projections from the spiral ganglion to the organ of Corti were not significantly different from wild type controls. Inner hair cell synapse formation also appeared unaffected in mature Chd7Gt/+ cochleae. However, histological analysis of adult Chd7Gt/+ cochleae revealed diminished satellite glial cells and hypermyelinated Type I spiral ganglion axons. We characterized the expression of CHD7 in developing inner ear glia and found CHD7 to be expressed during a tight window of inner ear development at the Schwann cell precursor stage at E9.5. While cochlear neurons appear to differentiate normally in the setting of Chd7 haploinsufficiency, our results suggest an important role for CHD7 in glial cells in the inner ear. This study highlights the dynamic nature of CHD7 activity during inner ear development in mice and contributes to understanding CHARGE syndrome pathology.

Development of a chronically-implanted mouse model for studies of cochlear health and implant function.

Mice with chronic cochlear implants can significantly contribute to our understanding of the relationship between cochlear health and implant function because of the availability of molecular tools for controlling conditions in the cochlea and transgenic lines modeling human disease. To date, research in implanted mice has mainly consisted of short-term studies, but since there are large changes in implant function following implant insertion trauma, and subsequent recovery in many cases, longer-term studies are needed to evaluate function and perception under stable conditions. Because frequent anesthetic administration can be especially problematic in mice, a chronic model that can be tested in the awake condition is desirable. Electrically-evoked compound action potentials (ECAPs) recorded with multichannel cochlear implants are useful functional measures because they can be obtained daily without anesthesia. In this study, we assessed changes and stability of ECAPs, electrically-evoked auditory brainstem responses (EABRs), ensemble spontaneous activity (ESA), and impedance data over time after implanting mice with multichannel implants. We then compared these data to histological findings in these implanted cochleae, and compared results from this chronic mouse model to data previously obtained in a well-established chronically-implanted guinea pig model. We determined that mice can be chronically implanted with cochlear implants, and ECAP recordings can be obtained frequently in an awake state for up to at least 42 days after implantation. These recordings can effectively monitor changes or stability in cochlear function over time. ECAP and EABR amplitude-growth functions (AGFs), AGF slopes, ESA levels and impedances in mice with multichannel implants appear similar to those found in guinea pigs with long-term multichannel implants. Animals with better survival of spiral ganglion neurons (SGNs) and inner hair cells (IHCs) have steeper AGF slopes, and larger ESA responses. The time course of post-surgical ear recovery may be quicker in mice and can show different patterns of recovery which seem to be dependent on the degree of insertion trauma and subsequent histological conditions. Histology showed varying degrees of cochlear damage with fibrosis present in all implanted mouse ears and small amounts of new bone in a few ears. Impedance changes over time varied within and across animals and may represent changes over time in multiple variables in the cochlear environment post-implantation. Due to the small size of the mouse, susceptibility to stress, and the higher potential for implant failure, chronic implantation in mice can be challenging, but overall is feasible and useful for cochlear implant research.

Scar Formation and Debris Elimination during Hair Cell Degeneration in the Adult DTR Mouse.

The auditory sensory epithelium of the mammalian inner ear is a highly organized structure that contains sensory hair cells (HCs) and non-sensory supporting cells (SCs). Following the partial loss of HCs after cochlear insults such as overstimulation or ototoxic drugs, SCs seal the luminal epithelial surface (reticular lamina) and reorganize its cellular pattern. Here we investigated the changes in the sensory epithelium following a rapid and severe cochlear insult in the diphtheria toxin receptor (DTR) mouse, where diphtheria toxin (DT) injection leads to a HC-specific lesion resulting in a complete HC loss. We found that DT-induced selective HC ablation could lead to a pattern of scar formation and apical cell-cell adherens and tight junction reorganization similar to that occurring after other types of cochlear insult. Prestin, an outer HC-specific protein, was present in amorphous clumps at the sites where SCs had expanded to fill the spaces vacated by the dead HCs for up to 2 months after the DT induced lesion. Many of the prestin clumps appeared to occupy spaces within SCs, suggesting that SCs participate in the removal process of HC corpses in the DTR deafness model. Prestin clumps could be seen in different areas all along the length of the SCs, and appeared to be inside the SCs as well as in the inter-cellular spaces between SCs. The findings suggest that HC elimination in the DTR deafness model follows a mechanism similar to that in overstimulation or ototoxicity models, making the DTR mouse useful for understanding the process underlying HC elimination and the role of SCs in this process.

GJB2 gene therapy and conditional deletion reveal developmental stage-dependent effects on inner ear structure and function.

Pathogenic variants in GJB2, the gene encoding connexin 26, are the most common cause of autosomal-recessive hereditary deafness. Despite this high prevalence, pathogenic mechanisms leading to GJB2-related deafness are not well understood, and cures are absent. Humans with GJB2-related deafness retain at least some auditory hair cells and neurons, and their deafness is usually stable. In contrast, mice with conditional loss of Gjb2 in supporting cells exhibit extensive loss of hair cells and neurons and rapidly progress to profound deafness, precluding the application of therapies that require intact cochlear cells. In an attempt to design a less severe Gjb2 animal model, we generated mice with inducible Sox10iCre ERT2 -mediated loss of Gjb2. Tamoxifen injection led to reduced connexin 26 expression and impaired function, but cochlear hair cells and neurons survived for 2 months, allowing phenotypic rescue attempts within this time. AAV-mediated gene transfer of GJB2 in mature mutant ears did not demonstrate threshold improvement and in some animals exacerbated hearing loss and resulted in hair cell loss. We conclude that Sox10iCre ERT2 ;Gjb2 flox/flox mice are valuable for studying the biology of connexin 26 in the cochlea. In particular, these mice may be useful for evaluating gene therapy vectors and development of therapies for GJB2-related deafness.

Combinatorial Atoh1 and Gfi1 induction enhances hair cell regeneration in the adult cochlea.

Mature mammalian cochlear hair cells (HCs) do not spontaneously regenerate once lost, leading to life-long hearing deficits. Attempts to induce HC regeneration in adult mammals have used over-expression of the HC-specific transcription factor Atoh1, but to date this approach has yielded low and variable efficiency of HC production. Gfi1 is a transcription factor important for HC development and survival. We evaluated the combinatorial effects of Atoh1 and Gfi1 over-expression on HC regeneration using gene transfer methods in neonatal cochlear explants, and in vivo in adult mice. Adenoviral over-expression of Atoh1 and Gfi1 in cultured neonatal cochlear explants resulted in numerous ectopic HC-like cells (HCLCs), with significantly more cells in Atoh1 + Gfi1 cultures than Atoh1 alone. In vitro, ectopic HCLCs emerged in regions medial to inner HCs as well as in the stria vascularis. In vivo experiments were performed in mature Pou4f3DTR mice in which HCs were completely and specifically ablated by administration of diphtheria toxin. Adenoviral expression of Atoh1 or Atoh1 + Gfi1 in cochlear supporting cells induced appearance of HCLCs, with Atoh1 + Gfi1 expression leading to 6.2-fold increase of new HCLCs after 4 weeks compared to Atoh1 alone. New HCLCs were detected throughout the cochlea, exhibited immature stereocilia and survived for at least 8 weeks. Combinatorial Atoh1 and Gfi1 induction is thus a promising strategy to promote HC regeneration in the mature mammalian cochlea.

Defects in vestibular sensory epithelia and innervation in mice with loss of Chd7 function: implications for human CHARGE syndrome.

CHD7 is a chromodomain gene mutated in CHARGE syndrome, a multiple anomaly condition characterized by ocular coloboma, heart defects, atresia of the choanae, retarded growth and development, genital hypoplasia, and ear defects including deafness and semicircular canal dysgenesis. Mice with heterozygous Chd7 deficiency have circling behavior and semicircular canal defects and are an excellent animal model for exploring the pathogenesis of CHARGE features. Inner ear vestibular defects have been characterized in heterozygous Chd7-deficient embryos and early postnatal mice, but it is not known whether vestibular defects persist throughout adulthood in Chd7-deficient mice or whether the vestibular sensory epithelia and their associated innervation and function are intact. Here we describe a detailed analysis of inner ear vestibular structures in mature mice that are heterozygous for a Chd7-deficient, gene-trapped allele (Chd7(Gt/+)). Chd7(Gt/+) mice display variable asymmetric lateral and posterior semicircular canal malformations, as well as defects in vestibular sensory epithelial innervation despite the presence of intact hair cells in the target organs. These observations have important functional implications for understanding the clinical manifestations of CHD7 mutations in humans and for designing therapies to treat inner ear vestibular dysfunction.

Whirler mutant hair cells have less severe pathology than shaker 2 or double mutants.

MYOSIN XV is a motor protein that interacts with the PDZ domain-containing protein WHIRLIN and transports WHIRLIN to the tips of the stereocilia. Shaker 2 (sh2) mice have a mutation in the motor domain of MYOSIN XV and exhibit congenital deafness and circling behavior, probably because of abnormally short stereocilia. Whirler (wi) mice have a similar phenotype caused by a deletion in the third PDZ domain of WHIRLIN. We compared the morphology of Whrn (wi/wi) and Myo15 (sh2/sh2) sensory hair cells and found that Myo15 (sh2/sh2) have more frequent pathology at the base of inner hair cells than Whrn (wi/wi), and shorter outer hair cell stereocilia. Considering the functional and morphologic similarities in the phenotypes caused by mutations in Myo15 and Whrn, and the physical interaction between their encoded proteins, we used a genetic approach to test for functional overlap. Double heterozygotes (Myo15 (sh2/+), Whrn (wi/+)) have normal hearing and no increase in hearing loss compared to normal littermates. Single and double mutants (Myo15 (sh2/sh2), Whrn (wi/wi)) exhibit abnormal persistence of kinocilia and microvilli, and develop abnormal cytoskeletal architecture. Double mutants are also similar to the single mutants in viability, circling behavior, and lack of a Preyer reflex. The morphology of cochlear hair cell stereocilia in double mutants reflects a dominance of the more severe Myo15 (sh2/sh2) phenotype over the Whrn (wi/wi) phenotype. This suggests that MYOSIN XV may interact with other proteins besides WHIRLIN that are important for hair cell maturation.

The role of bone morphogenetic protein 4 in inner ear development and function.

Bone Morphogenetic Protein 4 (BMP4) is a member of the TGF-beta superfamily and is known to be important for the normal development of many tissues and organs, including the inner ear. Bmp4 homozygous null mice die as embryos, but Bmp4 heterozygous null (Bmp4(+/-)) mice are viable and some adults exhibit a circling phenotype, suggestive of an inner ear defect. To understand the role of BMP4 in inner ear development and function, we have begun to study C57BL/6 Bmp4(+/-) mice. Quantitative testing of the vestibulo-collic reflex, which helps maintain head stability, demonstrated that Bmp4(+/-) mice that exhibit circling behavior have a poor response in the yaw axis, consistent with semicircular canal dysfunction. Although the hair cells of the ampullae were grossly normal, the stereocilia were greatly reduced in number. Auditory brainstem responses showed that Bmp4(+/-) mice have elevated hearing thresholds and immunohistochemical staining demonstrated decreased numbers of neuronal processes in the organ of Corti. Thus Bmp4(+/-) mice have structural and functional deficits in the inner ear.

Severe streptomycin ototoxicity in the mouse utricle leads to a flat epithelium but the peripheral neural degeneration is delayed.

The damaged vestibular sensory epithelium of mammals has a limited capacity for spontaneous hair cell regeneration, which largely depends on the transdifferentiation of surviving supporting cells. Little is known about the response of vestibular supporting cells to a severe insult. In the present study, we evaluated the impact of a severe ototoxic insult on the histology of utricular supporting cells and the changes in innervation that ensued. We infused a high dose of streptomycin into the mouse posterior semicircular canal to induce a severe lesion in the utricle. Both scanning electron microscopy and light microscopy of plastic sections showed replacement of the normal cytoarchitecture of the epithelial layer with a flat layer of cells in most of the samples. Immunofluorescence staining showed numerous cells in the severely damaged epithelial layer that were negative for hair cell and supporting cell markers. Nerve fibers under the flat epithelium had high density at the 1 month time point but very low density by 3 months. Similarly, the number of vestibular ganglion neurons was unchanged at 1 month after the lesion, but was significantly lower at 3 months. We therefore determined that the mouse utricular epithelium turns into a flat epithelium after a severe lesion, but the degeneration of neural components is slow, suggesting that treatments to restore balance by hair cell regeneration, stem cell therapy or vestibular prosthesis implantation will likely benefit from the short term preservation of the neural substrate.

p27(Kip1) deficiency causes organ of Corti pathology and hearing loss.

p27(Kip1) (p27) has been shown to inhibit several cyclin-dependent kinase molecules and to play a central role in regulating entry into the cell cycle. Once hair cells in the cochlea are formed, p27 is expressed in non-sensory cells of the organ of Corti and prevents their re-entry into the cell cycle. In one line of p27 deficient mice (p27(-/-)), cell division in the organ of Corti continues past its normal embryonic time, leading to continual production of cells in the organ of Corti. Here we report on the structure and function of the inner ear in another line of p27 deficient mice originating from the Memorial Sloan-Kettering Cancer Center. The deficiency in p27 expression of these mice is incomplete, as they retain expression of amino acids 52-197. We determined that mice homozygote for this mutation had severe hearing loss and their organ of Corti exhibited an increase in the number of inner and outer hair cells. There also was a marked increase in the number of supporting cells, with severe pathologies in pillar cells. These data show similarities between this p27(Kip1) mutation and another, previously reported null allele of this gene, and suggest that reducing the inhibition on the cell cycle in the organ of Corti leads to pathology and dysfunction. Manipulations to regulate the time and place of p27 inhibition will be necessary for inducing functionally useful hair cell regeneration.