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Inner ear hair cells convert the mechanical stimuli of sound, gravity, and head movement into electrical signals. This mechanotransduction process is initiated by opening of cation channels near the tips of hair cell stereocilia. Since the identity of these ion channels is unknown, and mutations in the gene encoding transmembrane channel-like 1 (TMC1) cause hearing loss without vestibular dysfunction in both mice and humans, we investigated the contribution of Tmc1 and the closely related Tmc2 to mechanotransduction in mice. We found that Tmc1 and Tmc2 were expressed in mouse vestibular and cochlear hair cells and that GFP-tagged TMC proteins localized near stereocilia tips. Tmc2 expression was transient in early postnatal mouse cochlear hair cells but persisted in vestibular hair cells. While mice with a targeted deletion of Tmc1 (Tmc1(Δ) mice) were deaf and those with a deletion of Tmc2 (Tmc2(Δ) mice) were phenotypically normal, Tmc1(Δ)Tmc2(Δ) mice had profound vestibular dysfunction, deafness, and structurally normal hair cells that lacked all mechanotransduction activity. Expression of either exogenous TMC1 or TMC2 rescued mechanotransduction in Tmc1(Δ)Tmc2(Δ) mutant hair cells. Our results indicate that TMC1 and TMC2 are necessary for hair cell mechanotransduction and may be integral components of the mechanotransduction complex. Our data also suggest that persistent TMC2 expression in vestibular hair cells may preserve vestibular function in humans with hearing loss caused by TMC1 mutations.

Presbycusis, or age-related hearing loss (ARHL), is a major public health issue. About half the phenotypic variance has been attributed to genetic factors. Here, we assessed the contribution to presbycusis of ultrarare pathogenic variants, considered indicative of Mendelian forms. We focused on severe presbycusis without environmental or comorbidity risk factors and studied multiplex family age-related hearing loss (mARHL) and simplex/sporadic age-related hearing loss (sARHL) cases and controls with normal hearing by whole-exome sequencing. Ultrarare variants (allele frequency [AF] < 0.0001) of 35 genes responsible for autosomal dominant early-onset forms of deafness, predicted to be pathogenic, were detected in 25.7% of mARHL and 22.7% of sARHL cases vs. 7.5% of controls (P = 0.001); half were previously unknown (AF < 0.000002). MYO6, MYO7A, PTPRQ, and TECTA variants were present in 8.9% of ARHL cases but less than 1% of controls. Evidence for a causal role of variants in presbycusis was provided by pathogenicity prediction programs, documented haploinsufficiency, three-dimensional structure/function analyses, cell biology experiments, and reported early effects. We also established Tmc1N321I/+ mice, carrying the TMC1:p.(Asn327Ile) variant detected in an mARHL case, as a mouse model for a monogenic form of presbycusis. Deafness gene variants can thus result in a continuum of auditory phenotypes. Our findings demonstrate that the genetics of presbycusis is shaped by not only well-studied polygenic risk factors of small effect size revealed by common variants but also, ultrarare variants likely resulting in monogenic forms, thereby paving the way for treatment with emerging inner ear gene therapy.

Defects of CIB2, calcium‐ and integrin‐binding protein 2, have been reported to cause isolated deafness, DFNB48 and Usher syndrome type‐IJ, characterized by congenital profound deafness, balance defects and blindness. We report here two new nonsense mutations (pGln12* and pTyr110*) in CIB2 patients displaying nonsyndromic profound hearing loss, with no evidence of vestibular or retinal dysfunction. Also, the generated CIB2 −/− mice display an early onset profound deafness and have normal balance and retinal functions. In these mice, the mechanoelectrical transduction currents are totally abolished in the auditory hair cells, whilst they remain unchanged in the vestibular hair cells. The hair bundle morphological abnormalities of CIB2 −/− mice, unlike those of mice defective for the other five known USH1 proteins, begin only after birth and lead to regression of the stereocilia and rapid hair‐cell death. This essential role of CIB2 in mechanotransduction and cell survival that, we show, is restricted to the cochlea, probably accounts for the presence in CIB2 −/− mice and CIB2 patients, unlike in Usher syndrome, of isolated hearing loss without balance and vision deficits. Synopsis A lack of any of the first five USH1 proteins in mice leads to structural hair bundle defects in the embryo, causing congenital profound deafness, and balance dysfunction. The lack of CIB2 both in mice and humans, however, reveals that CIB2 is critical for hearing but not for balance and vision. In addition to its key role in auditory mechanoelectrical transduction, CIB2 is required for postnatal maintenance of the structural integrity of the hair bundle and hair cell survival in the cochlea. A lack of CIB2 leads to the mislocalisation of integrins in stereocilia, revealing the importance of CIB2‐mediated interactions with the extracellular matrix for the correct shaping of the auditory hair bundles. A functional CIB2 is required for normal hearing, but not for balance and vision, in both mice and humans, as we found two novel nonsense mutations leading to nonsyndromic hearing loss without signs of retinitis pigmentosa or vestibular dysfunction. Graphical Abstract A lack of any of the first five USH1 proteins in mice leads to structural hair bundle defects in the embryo, causing congenital profound deafness, and balance dysfunction. The lack of CIB2 both in mice and humans, however, reveals that CIB2 is critical for hearing but not for balance and vision.

We study the mixing time of a random walker who moves inside a dynamical random cluster model on the d-dimensional torus of side-length n. In this model, edges switch at rate μ between open and closed, following a Glauber dynamics for the random cluster model with parameters p, q. At the same time, the walker jumps at rate 1 as a simple random walk on the torus, but is only allowed to traverse open edges. We show that for small enough p the mixing time of the random walker is of order n2/μ. In our proof we construct a non-Markovian coupling through a multi-scale analysis of the environment, which we believe could be more widely applicable.

Usher syndrome type III (USH3) is a genetic disorder characterized by progressive, post‐lingual hearing loss, variable vestibular dysfunction, and onset of retinitis pigmentosa. USH3 is caused by mutations in CLRN1, which encodes clarin‐1, a tetraspanin‐like protein. Mutations in CLRN2, which encodes the related protein clarin‐2, are also implicated in progressive, non‐syndromic hearing loss in both humans and mice. USH3 patients show considerable phenotypic variability, even among individuals with the same mutation. This variability may result from environmental factors or interactions with other inner ear genes, such as CLRN2. To investigate the functional interplay of these genes, we generated Clrn1–/−Clrn2−/− double knockout mice. RNA‐sequencing and functional/physiological analyses revealed that clarin‐1 and clarin‐2 jointly regulate mechanoelectrical transduction, ionic homeostasis, and synaptic organization. Their combined loss leads to more severe hearing phenotype compared to Clrn1−/− and Clrn2−/− mice, which reveals a functional compensation between them. CLRN2 variants may exacerbate hearing loss in USH3 patients, supporting inclusion of CLRN2 in genetic screening. By revealing a functional, compensatory interplay between clarin‐1 and clarin‐2, this study reframes CLRN1‐associated deafness as a network‐dependent disorder and provides a mechanistic basis for genetic stratification and therapeutic directions in USH3 and related sensorineural hearing loss. Functional compensation between clarin‐1 and clarin‐2 in cochlear hair cells. Hearing loss associated with CLRN1 mutations shows striking phenotypic variability; however, the underlying mechanisms remain poorly understood. This study reveals that clarin‐1 and clarin‐2 function cooperatively in cochlear hair cells to sustain mechanoelectrical transduction, ionic homeostasis, and synaptic stability. Partial compensation between the two proteins buffers auditory function, whereas their combined loss triggers severe auditory failure, redefining clarin‐associated deafness as a network‐dependent disorder.

Hearing relies on mechanically gated ion channels present in the actin‐rich stereocilia bundles at the apical surface of cochlear hair cells. Our knowledge of the mechanisms underlying the formation and maintenance of the sound‐receptive structure is limited. Utilizing a large‐scale forward genetic screen in mice, genome mapping and gene complementation tests, we identified Clrn2 as a new deafness gene. The Clrn2 clarinet / clarinet mice (p.Trp4* mutation) exhibit a progressive, early‐onset hearing loss, with no overt retinal deficits. Utilizing data from the UK Biobank study, we could show that CLRN2 is involved in human non‐syndromic progressive hearing loss. Our in‐depth morphological, molecular and functional investigations establish that while it is not required for initial formation of cochlear sensory hair cell stereocilia bundles, clarin‐2 is critical for maintaining normal bundle integrity and functioning. In the differentiating hair bundles, lack of clarin‐2 leads to loss of mechano‐electrical transduction, followed by selective progressive loss of the transducing stereocilia. Together, our findings demonstrate a key role for clarin‐2 in mammalian hearing, providing insights into the interplay between mechano‐electrical transduction and stereocilia maintenance. Synopsis The study identifies CLRN2 as a new deafness gene required for maintenance of transducing stereocilia in the sensory cochlear hair cells. Lack of clarin‐2 leads to an early‐onset hearing loss in mice. CLRN2 is associated with human non‐syndromic progressive hearing loss. Utilizing an unbiased forward genetic screen in mice, a Clrn2 gene nonsense mutation (p.Trp4*) was identified as the cause of deafness in the clarinet mouse mutant ( Clrn2 clarinet ). Analysis of data from the UK Biobank study identifies CLRN2 as a novel candidate gene for human non‐syndromic progressive age‐related hearing loss. While clarin‐2 is non‐essential for stereocilia bundle patterning, it is indispensable for their maintenance, with loss of clarin‐2 leading to stereocilia resorption, decreased mechano‐electrical transduction, and progressive hearing impairment. Graphical Abstract The study identifies Clarin‐2 as a new deafness gene required for maintenance of transducing stereocilia in the sensory cochlear hair cells. Lack of clarin‐2 leads to an early‐onset hearing loss in mice. CLRN2 is associated with human non‐syndromic progressive hearing loss.

Deafness is the most common form of sensory impairment in humans and is frequently caused by single gene mutations. Interestingly, different mutations in a gene can cause syndromic and nonsyndromic forms of deafness, as well as progressive and age-related hearing loss. We provide here an explanation for the phenotypic variability associated with mutations in the cadherin 23 gene (CDH23). CDH23 null alleles cause deaf-blindness (Usher syndrome type 1D; USH1D), whereas missense mutations cause nonsyndromic deafness (DFNB12). In a forward genetic screen, we have identified salsa mice, which suffer from hearing loss due to a Cdh23 missense mutation modeling DFNB12. In contrast to waltzer mice, which carry a CDH23 null allele mimicking USH1D, hair cell development is unaffected in salsa mice. Instead, tip links, which are thought to gate mechanotransduction channels in hair cells, are progressively lost. Our findings suggest that DFNB12 belongs to a new class of disorder that is caused by defects in tip links. We propose that mutations in other genes that cause USH1 and nonsyndromic deafness may also have distinct effects on hair cell development and function.

The molecular composition of the hair cell transduction channel has not been identified. Here we explore the novel hypothesis that hair cell transduction channels include HCN subunits. The HCN family of ion channels includes four members, HCN1-4. They were originally identified as the molecular correlates of the hyperpolarization-activated, cyclic nucleotide gated ion channels that carry currents known as If, IQ or Ih. However, based on recent evidence it has been suggested that HCN subunits may also be components of the elusive hair cell transduction channel. To investigate this hypothesis we examined expression of mRNA that encodes HCN1-4 in sensory epithelia of the mouse inner ear, immunolocalization of HCN subunits 1, 2 and 4, uptake of the transduction channel permeable dye, FM1-43 and electrophysiological measurement of mechanotransduction current. Dye uptake and transduction current were assayed in cochlear and vestibular hair cells of wildtype mice exposed to HCN channel blockers or a dominant-negative form of HCN2 that contained a pore mutation and in mutant mice that lacked HCN1, HCN2 or both. We found robust expression of HCNs 1, 2 and 4 but little evidence that localized HCN subunits in hair bundles, the site of mechanotransduction. Although high concentrations of the HCN antagonist, ZD7288, blocked 50-70% of the transduction current, we found no reduction of transduction current in either cochlear or vestibular hair cells of HCN1- or HCN2- deficient mice relative to wild-type mice. Furthermore, mice that lacked both HCN1 and HCN2 also had normal transduction currents. Lastly, we found that mice exposed to the dominant-negative mutant form of HCN2 had normal transduction currents as well. Taken together, the evidence suggests that HCN subunits are not required for mechanotransduction in hair cells of the mouse inner ear.

Our understanding of the mechanisms underlying inherited forms of inner ear deficits has considerably improved during the past 20 y, but we are still far from curative treatments. We investigated gene replacement as a strategy for restoring inner ear functions in a mouse model of Usher syndrome type 1G, characterized by congenital profound deafness and balance disorders. These mice lack the scaffold protein sans, which is involved both in the morphogenesis of the stereociliary bundle, the sensory antenna of inner ear hair cells, and in the mechanoelectrical transduction process. We show that a single delivery of the sans cDNA by the adenoassociated virus 8 to the inner ear of newborn mutant mice reestablishes the expression and targeting of the protein to the tips of stereocilia. The therapeutic gene restores the architecture and mechanosensitivity of stereociliary bundles, improves hearing thresholds, and durably rescues these mice from the balance defects. Our results open up new perspectives for efficient gene therapy of cochlear and vestibular disorders by showing that even severe dysmorphogenesis of stereociliary bundles can be corrected.

This thesis deals with Random Walks on graphs that change over time in a random manner, more precisely we analyse Random Walks on Dynamical Percolation. In this model, the edges of a graph G are either open or closed and refresh their status at rate μ independently from all other edges, while at the same time a random walker moves on G at rate 1 but only along edges which are open. In Chapter 3 we present the known results about mixing time for random walks in dynamical percolation and we give a sketch of the proof of the upper bound for the mixing time of Random Walk on Dynamical Percolation when Gn = T_d^n for all p < p_c discussed in detail in [PSS15]. Later, we show a bound on the mixing time of the Random-Cluster model for lattices with polynomial growth. Finally, we introduce Random Walks on Dynamical Random Cluster. This model is similar to the Random Walks on Dynamical Percolation with the only difference that the refresh of the edges depends on the configuration of the open edges in the graph at the time of the update. We prove that on the d-dimensional torus with side length n, in the subcritical regime, the mixing time for the full system is bounded above by n^2/μ up to constants.