The inflammatory foreign body response (FBR) following cochlear implantation (CI) can negatively impact CI outcomes, including increased electrode impedances. This study aims to investigate the long-term efficacy of dexamethasone-eluting cochlear implant and locally delivered dexamethasone, a potent anti-inflammatory glucocorticoid, on the intracochlear FBR and electrical impedance post-implantation in a murine model. Preliminary impedance data in humans are also provided as a complement to the murine data to illustrate generalizability and reinforce implications related to clinical application. The left ears of CX3CR1+/GFP Thy1+/YFP (macrophage-neuron dual reporter) mice were implanted with dexamethasone-eluting cochlear implants (Dex-CI) or standard implant (Standard-CI) while the right ear served as unoperated control. Another group of dual reporter mice was implanted with a standard CI electrode array followed by injection of dexamethasone in the middle ear to mimic current clinical practice (Dex-local). Mouse implants were electrically stimulated with serial measurements of electrical impedance. Human subjects were implanted with either standard or Dex-CI followed by serial impedance measurements. Dex-CI reduced electrical impedance in the murine model and human subjects and inflammatory FBR in the murine model for an extended period. Dex-local in the murine model is ineffective for long-term reduction of FBR and electrode impedance. Our data suggests that dexamethasone-eluting arrays are more effective than the current clinical practice of locally applied dexamethasone in reducing FBR and electrical impedance.

Stereocilia are rod-like mechanosensory projections consisting of unidirectionally oriented actin filaments that extend into the inner ear hair cell cytoskeleton, forming dense rootlets. Taperin (TPRN) localizes to the narrowed-down base of stereocilia, where they pivot in response to sound and gravity. We show that TPRN-deficient mice have progressive deafness characterized by gradual asynchronous retraction and fusion of outer and inner hair cell stereocilia, followed by synaptic abnormalities. Stereocilia that lack TPRN develop warped rootlets with gradual loss of TRIOBP-5 and ANKRD24 from mechanosensory rows starting postnatally. In contrast, TPRN overexpression causes excessive F-actin bundling, extra rows, and over-elongation of stereocilia during development. Purified full-length mouse TPRN cross-links F-actin into bendable bundles reflecting in vivo data. This F-actin-bundling ability is attributed to the TPRN N-terminal region. TPRN interacts with the membrane receptor PTPRQ, connecting the F-actin core to the plasma membrane, stabilizing stereocilia. Thus, TPRN is a specialized F-actin bundler strategically located to augment stereocilia rootlet formation and their pivot point flexibility for sustained sound-induced deflections.

The stria vascularis (SV) is an essential component of the inner ear that regulates the ionic environment required for hearing. SV degeneration disrupts cochlear homeostasis, leading to irreversible hearing loss, yet a comprehensive understanding of the SV, and consequently therapeutic availability for SV degeneration, is lacking. We developed a whole-tissue explant model from neonatal and mature mice to create a platform for advancing SV research. We validated our model by demonstrating that the proliferative behavior of the SV in vitro mimics SV in vivo. We also provided evidence for pharmacological experimentation by investigating the role of Wnt/b-catenin signaling in SV proliferation. Finally, we performed single-cell RNA sequencing from in vivo neonatal and mature mouse SV and surrounding tissue and revealed key genes and pathways that may play a role in SV proliferation and maintenance. Together, our results contribute new insights into investigating biological solutions for SV-associated hearing loss.

Deafness in vertebrates is associated with variants of hundreds of genes. Yet, many mutant genes causing rare forms of deafness remain to be discovered. A consanguineous Pakistani family segregating nonsyndromic deafness in two sibships were studied using microarrays and exome sequencing. A 1.2 Mb locus (DFNB128) on chromosome 5q11.2 encompassing six genes was identified. In one of the two sibships of this family, a novel homozygous recessive variant NM_005921.2:c.4460G>A p.(Arg1487His) in the kinase domain of MAP3K1 co-segregated with nonsyndromic deafness. There are two previously reported Map3k1-kinase-deficient mouse models that are associated with recessively inherited syndromic deafness. MAP3K1 phosphorylates serine and threonine and functions in a signaling pathway where pathogenic variants of HGF, MET, and GAB1 were previously reported to be associated with human deafness DFNB39, DFNB97, and DFNB26, respectively. Our single-cell transcriptome data of mouse cochlea mRNA show expression of Map3k1 and its signaling partners in several inner ear cell types suggesting a requirement of wild-type MAP3K1 for normal hearing. In contrast to dominant variants of MAP3K1 associated with Disorders of Sex Development 46,XY sex-reversal, our computational modeling of the recessive substitution p.(Arg1487His) predicts a subtle structural alteration in MAP3K1, consistent with the limited phenotype of nonsyndromic deafness.

SummaryThe stria vascularis (SV), part of the blood-labyrinth barrier, is an essential component of the inner ear that regulates the ionic environment required for hearing. SV degeneration disrupts cochlear homeostasis, leading to irreversible hearing loss, yet a comprehensive understanding of the SV, and consequently therapeutic availability for SV degeneration, is lacking. We developed a whole-tissue explant model from neonatal and adult mice to create a robust platform for SV research. We validated our model by demonstrating that the proliferative behaviour of the SVin vitromimics SVin vivo,providing a representative model and advancing high-throughput SV research. We also provided evidence for pharmacological intervention in our system by investigating the role of Wnt/β-catenin signaling in SV proliferation. Finally, we performed single-cell RNA sequencing fromin vivoneonatal and adult mouse SV and revealed key genes and pathways that may play a role in SV proliferation and maintenance. Together, our results contribute new insights into investigating biological solutions for SV-associated hearing loss.SignificanceHearing loss impairs our ability to communicate with people and interact with our environment. This can lead to social isolation, depression, cognitive deficits, and dementia. Inner ear degeneration is a primary cause of hearing loss, and our study provides an in depth look at one of the major sites of inner ear degeneration: the stria vascularis. The stria vascularis and associated blood-labyrinth barrier maintain the functional integrity of the auditory system, yet it is relatively understudied. By developing a newin vitromodel for the young and adult stria vascularis and using single cell RNA sequencing, our study provides a novel approach to studying this tissue, contributing new insights and widespread implications for auditory neuroscience and regenerative medicine.Highlights- We established an organotypic explant system of the neonatal and adult stria vascularis with an intact blood-labyrinth barrier.- Proliferation of the stria vascularis decreases with agein vitro, modelling its proliferative behaviourin vivo.- Pharmacological studies using ourin vitroSV model open possibilities for testing injury paradigms and therapeutic interventions.- Inhibition of Wnt signalling decreases proliferation in neonatal stria vascularis.- We identified key genes and transcription factors unique to developing and mature SV cell types using single cell RNA sequencing.

The inflammatory foreign body response (FBR) following cochlear implantation (CI) can negatively impact CI outcomes, including increased electrode impedances. This study aims to investigate the long-term efficacy of dexamethasone eluting cochlear implant and locally delivered dexamethasone, a potent anti-inflammatory glucocorticoid on the intracochlear FBR and electrical impedance post-implantation in a murine model and human subjects. The left ears of CX3CR1 +/GFP Thy1 +/YFP (macrophage-neuron dual reporter) mice were implanted with dexamethasone-eluting cochlear implants (Dex-CI) or standard implant (Standard-CI) while the right ear served as unoperated control. Another group of dual reporter mice was implanted with a standard CI electrode array followed by injection of dexamethasone in the middle ear to mimic current clinical practice (Dex-local). Mouse implants were electrically stimulated with serial measurement of electrical impedance. Human subjects were implanted with either standard or Dex-CI followed by serial impedance measurements. Dex-CI reduced electrical impedance in the murine model and human subjects and inflammatory FBR in the murine model for an extended period. Dex-local in the murine model is ineffective for long-term reduction of FBR and electrode impedance. Our data suggest that dexamethasone eluting arrays are more effective than the current clinical practice of locally applied dexamethasone in reducing FBR and electrical impedance.

The stria vascularis (SV) is a stratified epithelium in the lateral wall of the mammalian cochlea, responsible for both endolymphatic ion homeostasis and generation of the endocochlear potential (EP) critical for normal hearing. The SV has three layers consisting predominantly of basal, intermediate, and marginal cells. Intermediate and marginal cells form an intricate interdigitated network of cell projections making discrimination of the cells challenging. To enable intermediate cell visualization, we engineered by BAC transgenesis, reporter mouse lines expressing ZsGreen fluorescent protein under the control of Kcnj10 promoter and regulatory sequences. Kcnj10 encodes KCNJ10 protein (also known as Kir4.1 or Kir1.2), an ATP-sensitive inwardly-rectifying potassium channel critical to EP generation, highly expressed in SV intermediate cells. In these transgenic mice, ZsGreen fluorescence mimics Kcnj10 endogenous expression in the cochlea and was detected in the intermediate cells of the SV, in the inner phalangeal cells, Hensen's, Deiters' and pillar cells, in a subset of spiral ganglion neurons, and in glial cells. We show that expression of the transgene in hemizygous mice does not alter auditory function, nor EP These transgenic Tg(Kcnj10-ZsGreen) mice allow live and fixed tissue visualization of ZsGreen-expressing intermediate cells and will facilitate future studies of stria vascularis cell function.

Objective: To find a more objective method of assessing which patients should be screened for a vestibular schwannoma (VS) with magnetic resonance imaging (MRI) using a deep-learning algorithm to assess clinical and audiometric data. Materials and Methods: Clinical and audiometric data were collected for 592 patients who received an audiogram between January 2015 and 2020 at Duke University Health Center with and without VS confirmed by MRI. These data were analyzed using a deep learning-based analysis to determine if the need for MRI screening could be assessed more objectively with adequate sensitivity and specificity. Results: Patients with VS showed slightly elevated, but not statistically significant, mean thresholds compared to those without. Tinnitus, gradual hearing loss, and aural fullness were more common in patients with VS. Of these, only the presence of tinnitus was statistically significant. Several machine learning algorithms were used to incorporate and model the collected clinical and audiometric data, but none were able to distinguish ears with and without confirmed VS. When tumor size was taken into account the analysis was still unable to distinguish a difference. Conclusions: Using audiometric and clinical data, deep learning-based analyses failed to produce an adequately sensitive and specific model for the detection of patients with VS. This suggests that a specific pattern of audiometric asymmetry and clinical symptoms may not necessarily be predictive of the presence/absence of VS to a level that clinicians would be comfortable forgoing an MRI.

Hereditary deafness and retinal dystrophy are each genetically heterogenous and clinically variable. Three small unrelated families segregating the combination of deafness and retinal dystrophy were studied by exome sequencing (ES). The proband of Family 1 was found to be compound heterozygous for NM_004525.3: LRP2: c.5005A > G, p.(Asn1669Asp) and c.149C > G, p.(Thr50Ser). In Family 2, two sisters were found to be compound heterozygous for LRP2 variants, p.(Tyr3933Cys) and an experimentally confirmed c.7715 + 3A > T consensus splice-altering variant. In Family 3, the proband is compound heterozygous for a consensus donor splice site variant LRP2: c.8452_8452 + 1del and p.(Cys3150Tyr). In mouse cochlea, Lrp2 is expressed abundantly in the stria vascularis marginal cells demonstrated by smFISH, single-cell and single-nucleus RNAseq, suggesting that a deficiency of LRP2 may compromise the endocochlear potential, which is required for hearing. LRP2 variants have been associated with Donnai-Barrow syndrome and other multisystem pleiotropic phenotypes different from the phenotypes of the four cases reported herein. Our data expand the phenotypic spectrum associated with pathogenic variants in LRP2 warranting their consideration in individuals with a combination of hereditary hearing loss and retinal dystrophy.

Abstract Deafness in vertebrates is associated with variants of hundreds of genes. Yet, many genes causing rare forms of deafness remain to be discovered. Microarrays and exome sequencing were used to study a consanguineous Pakistani family segregating nonsyndromic deafness in two sibships. A 1.2 Mb locus (DFNB110) on chromosome 5q11.2 encompassing six genes was identified. In one of the two sibships of this family, a novel homozygous recessive variant NM_005921.2:c.4460G>A p.(Arg1487His) in the kinase domain of MAP3K1 co-segregated with nonsyndromic deafness. MAP3K1 phosphorylates serine and threonine or tyrosine and functions in a signaling pathway where pathogenic variants of HGF, MET and GAB1 were previously reported to be associated with human deafness DFNB39, DFNB97 and DFNB26, respectively. Single-cell transcriptome data obtained from mouse cochlea mRNA show Map3k1 expression and its associated signaling partners necessary for hearing. Computational modeling of p.(Arg1487His) predicts a subtle structural alteration consistent with the limited DFNB110 phenotype in contrast to the pleiotropic phenotype of dominant MAP3K1 variants causing Disorders of Sex Development 46,XY sex-reversal and the reported syndromic deafness phenotype of two different protein truncating recessive variants of mouse Map3k1.

Abstract Deafness in vertebrates is associated with variants of hundreds of genes. Yet, many genes causing rare forms of deafness remain to be discovered. Microarrays and exome sequencing were used to study a consanguineous Pakistani family segregating nonsyndromic deafness in two sibships. A 1.2 Mb locus ( DFNB110 ) on chromosome 5q11.2 encompassing six genes was identified. In one of the two sibships of this family, a novel homozygous recessive variant NM_005921.2:c.4460G>A p.(Arg1487His) in the kinase domain of MAP3K1 co-segregated with nonsyndromic deafness. MAP3K1 phosphorylates serine and threonine or tyrosine and functions in a signaling pathway where pathogenic variants of HGF , MET and GAB1 were previously reported to be associated with human deafness DFNB39, DFNB97 and DFNB26, respectively. Single-cell transcriptome data obtained from mouse cochlea mRNA show Map3k1 expression and its associated signaling partners necessary for hearing. Computational modeling of p.(Arg1487His) predicts a subtle structural alteration consistent with the limited DFNB110 phenotype in contrast to the pleiotropic phenotype of dominant MAP3K1 variants causing Disorders of Sex Development 46,XY sex-reversal and the reported syndromic deafness phenotype of two different protein truncating recessive variants of mouse Map3k1 .

Endocochlear potential, which is generated by the stria vascularis, is essential to maintain an environment conducive to appropriate hair cell mechanotransduction and ultimately hearing. Pathologies of the stria vascularis can result in a decreased hearing. Dissection of the adult stria vascularis allows for focused single-nucleus capture and subsequent single-nucleus sequencing and immunostaining. These techniques are used to study stria vascularis pathophysiology at the single-cell level.Single-nucleus sequencing can be used in the setting of transcriptional analysis of the stria vascularis. Meanwhile, immunostaining continues to be useful in identifying specific populations of cells. Both methods require proper stria vascularis dissection as a prerequisite, which can prove to be technically challenging.

Hypothesis: Proteins enriched in the perilymph proteome of Meńier̀e disease (MD) patients may identify affected cell types. Utilizing single-cell transcriptome datasets from the mammalian cochlea, we hypothesize that these enriched perilymph proteins can be localized to specific cochlear cell types. Background: The limited understanding of human inner ear pathologies and their associated biomolecular variations hinder efforts to develop disease-specific diagnostics and therapeutics. Perilymph sampling and analysis is now enabling further characterization of the cochlear microenvironment. Recently, enriched inner ear protein expression has been demonstrated in patients with MD compared to patients with other inner ear diseases. Localizing expression of these proteins to cochlear cell types can further our knowledge of potential disease pathways and subsequent development of targeted therapeutics. Methods: We compiled previously published data regarding differential perilymph proteome profiles amongst patients with MD, otosclerosis, enlarged vestibular aqueduct, sudden hearing loss, and hearing loss of undefined etiology (controls). Enriched proteins in MD were cross-referenced against published single-cell/single-nucleus RNA-sequencing datasets to localize gene expression to specific cochlear cell types. Results: In silico analysis of single-cell transcriptomic datasets demonstrates enrichment of a unique group of perilymph proteins associated with MD in a variety of intracochlear cells, and some exogeneous hematologic and immune effector cells. This suggests that these cell types may play an important role in the pathology associated with late MD, suggesting potential future areas of investigation for MD pathophysiology and treatment. Conclusions: Perilymph proteins enriched in MD are expressed by specific cochlear cell types based on in silico localization, potentially facilitating development of disease-specific diagnostic markers and therapeutics.

Corticosteroids, oral or transtympanic, remain the mainstay for inner ear diseases characterized by hearing fluctuation or sudden changes in hearing, including sudden sensorineural hearing loss (SSNHL), Meniere’s disease (MD), and autoimmune inner ear disease (AIED). Despite their use across these diseases, the rate of complete recovery remains low, and results across the literature demonstrates significant heterogeneity with respect to the effect of corticosteroids, suggesting a need to identify more efficacious treatment options. Previously, our group has cross-referenced steroid-responsive genes in the cochlea with published single-cell and single-nucleus transcriptome datasets to demonstrate that steroid-responsive differentially regulated genes are expressed in spiral ganglion neurons (SGN) and stria vascularis (SV) cell types. These differentially regulated genes represent potential druggable gene targets. We utilized multiple gene target databases (DrugBank, Pharos, and LINCS) to identify orally administered, FDA approved medications that potentially target these genes. We identified 42 candidate drugs that have been shown to interact with these genes, with an emphasis on safety profile, and tolerability. This study utilizes multiple databases to identify drugs that can target a number of druggable genes in otologic disorders that are commonly treated with steroids, providing a basis for establishing novel repurposing treatment trials.

Background Treatment of many types of hearing instability in humans, including sudden sensorineural hearing loss, Meniere's disease, and autoimmune inner ear disease, rely heavily on the utilization of corticosteroids delivered both by oral and transtympanic routes. Despite this use, there is heterogeneity in the response to treatment with corticosteroids in humans with these diseases. The mechanisms by which corticosteroids exert their effect and the cell types in which they exert their effects in the inner ear remain poorly characterized. In this study, we localize steroid-responsive genes to cochlear cell types using previously published transcriptome datasets from the mammalian cochlea. Methods Steroid-responsive genes were localized to specific cochlear cell types using existing transcriptome datasets from wild-type mammalian cochlea exposed to systemic and transtympanic steroids, as well as previously published single-cell and single-nucleus RNA-sequencing datasets from the mammalian cochlea. Gene ontology (GO) analysis of differentially expressed genes (DEGs) was performed using PANTHER to investigate cellular processes implicated in transtympanic vs. systemic steroid action in the cochlea. Results Steroid-responsive genes were localized to specific cell types and regions in the cochlea including the stria vascularis, organ of Corti, and spiral ganglion neurons (SGN). Analyses demonstrate differential prevalence of steroid-responsive genes. GO analysis demonstrated steroid-responsive DEGs in the SGN to be associated with angiogenesis, apoptosis, and cytokine-mediated anti-inflammatory pathways. Conclusions Single-cell and single-nucleus transcriptome datasets localize steroid-responsive genes to specific regions in the cochlea. Further study of these regionally-specific steroid-responsive genes may provide insight into the mechanisms of and clinical response to corticosteroids in diseases of hearing instability.

Objective: To identify genes implicated in sudden sensorineural hearing loss (SSNHL) and localize their expression in the cochlea to further explore potential pathogenic mechanisms and therapeutic targets. Study Design: Systematic literature review and bioinformatics analysis. Data Sources: The following sources were searched from inception through July 2, 2020: PubMed-NCBI, MEDLINE, Embase, CINAHL, Cochrane Library, ClinicalTrials.gov, OpenGrey, GreyNet, GreyLiterature Report, and European Union Clinical Trials Registry. PubMed-NCBI and MEDLINE were additionally searched for human temporal bone histopathologic studies related to SSNHL. Methods: Literature review of candidate SSNHL genes was conducted according to PRISMA guidelines. Existing temporal bone studies from SSNHL patients were analyzed to identify the most commonly affected inner ear structures. Previously published single-cell and single-nucleus RNA-Seq datasets of the adult mouse stria vascularis, as well as postnatal day 7 and 15 mouse cochlear hair cells and supporting cells, were utilized for localization of the SSNHL-related genes curated through literature review. Conclusions: We report 92 unique single nucleotide polymorphisms (SNPs) in 76 different genes that have been investigated in relation to SSNHL in the literature. We demonstrate that a subset of these genes are expressed by cell types in the adult mouse stria vascularis and organ of Corti, consistent with findings from temporal bone studies in human subjects with SSNHL. We highlight several potential genetic targets relevant to current and possible future SSNHL treatments.

Hearing loss and impaired fertility are common human disorders each with multiple genetic causes. Sometimes deafness and impaired fertility, which are the hallmarks of Perrault syndrome, co-occur in a person. Perrault syndrome is inherited as an autosomal recessive disorder characterized by bilateral mild to severe childhood sensorineural hearing loss with variable age of onset in both sexes and ovarian dysfunction in females who have a 46, XX karyotype. Since the initial clinical description of Perrault syndrome 70 years ago, the phenotype of some subjects may additionally involve developmental delay, intellectual deficit and other neurological disabilities, which can vary in severity in part dependent upon the genetic variants and the gene involved. Here, we review the molecular genetics and clinical phenotype of Perrault syndrome and focus on supporting evidence for the eight genes (CLPP, ERAL1, GGPS1, HARS2, HSD17B4, LARS2, RMND1, TWNK) associated with Perrault syndrome. Variants of these eight genes only account for approximately half of the individuals with clinical features of Perrault syndrome where the molecular genetic base remains under investigation. Additional environmental etiologies and novel Perrault disease-associated genes remain to be identified to account for unresolved cases. We also report a new genetic variant of CLPP, computational structural insight about CLPP and single cell RNAseq data for eight reported Perrault syndrome genes suggesting a common cellular pathophysiology for this disorder. Some unanswered questions are raised to kindle future research about Perrault syndrome.

Hearing loss is a problem that impacts a significant proportion of the adult population. Cochlear hair cell (HC) loss due to loud noise, chemotherapy and aging is the major underlying cause. A significant proportion of these individuals are dissatisfied with available treatment options which include hearing aids and cochlear implants. An alternative approach to restore hearing would be to regenerate HCs. Such therapy would require a recapitulation of the complex architecture of the organ of Corti, necessitating regeneration of both mature HCs and supporting cells (SCs). Transcriptional profiles of the mature cell types in the cochlea are necessary to can provide a metric for eventual regeneration therapies. To assist in this effort, we sought to provide the first single-cell characterization of the adult cochlear SC transcriptome. We performed single-cell RNA-Seq on FACS-purified adult cochlear SCs from the Lfng(EGFP) adult mouse, in which SCs express GFP. We demonstrate that adult cochlear SCs are transcriptionally distinct from their perinatal counterparts. We establish cell-type-specific adult cochlear SC transcriptome profiles, and we validate these expression profiles through a combination of both fluorescent immunohistochemistry and in situ hybridization co-localization and quantitative polymerase chain reaction (qPCR) of adult cochlear SCs. Furthermore, we demonstrate the relevance of these profiles to the adult human cochlea through immunofluorescent human temporal bone histopathology. Finally, we demonstrate cell cycle regulator expression in adult SCs and perform pathway analyses to identify potential mechanisms for facilitating mitotic regeneration (cell proliferation, differentiation, and eventually regeneration) in the adult mammalian cochlea. Our findings demonstrate the importance of characterizing mature as opposed to perinatal SCs.

The stria vascularis generates the endocochlear potential and is involved in processes that underlie ionic homeostasis in the cochlear endolymph, both which play essential roles in hearing. The histological hallmark of Meniere's disease (MD) is endolymphatic hydrops, which refers to the bulging or expansion of the scala media, which is the endolymph-containing compartment of the cochlea. This histologic hallmark suggests that processes that disrupt ion homeostasis or potentially endocochlear potential may underlie MD. While treatments exist for vestibular symptoms related to MD, effective therapies for hearing fluctuation and hearing loss seen in MD remain elusive. Understanding the potential cell types involved in MD may inform the creation of disease mouse models and provide insight into underlying mechanisms and potential therapeutic targets. For these reasons, we compare published datasets related to MD in humans with our previously published adult mouse stria vascularis single-cell and single-nucleus RNA-Seq datasets to implicate potentially involved stria vascularis (SV) cell types in MD. Finally, we provide support for these implicated cell types by demonstrating co-expression of select candidate genes for MD within SV cell types.

[This corrects the article DOI: 10.3389/fnmol.2020.00013.].