Bifeng Pan

TMC1 and TMC2 Are Components of the Mechanotransduction Channel in Hair Cells of the Mammalian Inner Ear

Sensory transduction in auditory and vestibular hair cells requires expression of transmembrane channel-like (Tmc) 1 and 2 genes, but the function of these genes is unknown. To investigate the hypothesis that TMC1 and TMC2 proteins are components of the mechanosensitive ion channels that convert mechanical information into electrical signals, we recorded whole-cell and single-channel currents from mouse hair cells that expressed Tmc1, Tmc2, or mutant Tmc1. Cells that expressed Tmc2 had high calcium permeability and large single-channel currents, while cells with mutant Tmc1 had reduced calcium permeability and reduced single-channel currents. Cells that expressed Tmc1 and Tmc2 had a broad range of single-channel currents, suggesting multiple heteromeric assemblies of TMC subunits. The data demonstrate TMC1 and TMC2 are components of hair cell transduction channels and contribute to permeation properties. Gradients in TMC channel composition may also contribute to variation in sensory transduction along the tonotopic axis of the mammalian cochlea.

Tmc gene therapy restores auditory function in deaf mice

Injection of AAV vectors that encoded wild-type Tmc1 or Tmc2 restores auditory function in mouse models of human deafnesses DFNB7/11 and DFNA36. Can you hear me now? Because genetics is a major cause of deafness, Askew and colleagues developed a therapeutic approach to replace mutant genes associated with the mechanotransduction machinery of the inner ear. The gene encoding transmembrane channel–like 1, Tmc1, or its ortholog, Tmc2, was packaged in adeno-associated viral vectors and delivered to mice with mutations in Tmc1 or Beethoven—models representative of autosomal recessive and dominant human deafness, respectively. Both vectors were able to transduce inner hair cells of the mouse cochlea, and partially restore hearing, as determined by auditory brainstem responses and startle reflexes. More than 30 TMC1 mutations have been implicated in recessive prelingual deafness, so it is hoped that this gene therapeutic approach will work long-term to maintain hearing recovery, perhaps supplementary existing technologies such as cochlear implants and hearing aids. Genetic hearing loss accounts for up to 50% of prelingual deafness worldwide, yet there are no biologic treatments currently available. To investigate gene therapy as a potential biologic strategy for restoration of auditory function in patients with genetic hearing loss, we tested a gene augmentation approach in mouse models of genetic deafness. We focused on DFNB7/11 and DFNA36, which are autosomal recessive and dominant deafnesses, respectively, caused by mutations in transmembrane channel–like 1 (TMC1). Mice that carry targeted deletion of Tmc1 or a dominant Tmc1 point mutation, known as Beethoven, are good models for human DFNB7/11 and DFNA36. We screened several adeno-associated viral (AAV) serotypes and promoters and identified AAV2/1 and the chicken β-actin (Cba) promoter as an efficient combination for driving the expression of exogenous Tmc1 in inner hair cells in vivo. Exogenous Tmc1 or its closely related ortholog, Tmc2, were capable of restoring sensory transduction, auditory brainstem responses, and acoustic startle reflexes in otherwise deaf mice, suggesting that gene augmentation with Tmc1 or Tmc2 is well suited for further development as a strategy for restoration of auditory function in deaf patients who carry TMC1 mutations.

Small molecule activators of TRPML3

We conducted a high-throughput screen for small molecule activators of the TRPML3 ion channel, which, when mutated, causes deafness and pigmentation defects. Cheminformatics analyses of the 53 identified and confirmed compounds revealed nine different chemical scaffolds and 20 singletons. We found that agonists strongly potentiated TRPML3 activation with low extracytosolic [Na(+)]. This synergism revealed the existence of distinct and cooperative activation mechanisms and a wide dynamic range of TRPML3 activity. Testing compounds on TRPML3-expressing sensory hair cells revealed the absence of activator-responsive channels. Epidermal melanocytes showed only weak or no responses to the compounds. These results suggest that TRPML3 in native cells might be absent from the plasma membrane or that the protein is a subunit of heteromeric channels that are nonresponsive to the activators identified in this screen.

TMC1 and TMC2 Localize at the Site of Mechanotransduction in Mammalian Inner Ear Hair Cell Stereocilia.

Mechanosensitive ion channels at stereocilia tips mediate mechanoelectrical transduction (MET) in inner ear sensory hair cells. Transmembrane channel-like 1 and 2 (TMC1 and TMC2) are essential for MET and are hypothesized to be components of the MET complex, but evidence for their predicted spatiotemporal localization in stereocilia is lacking. Here, we determine the stereocilia localization of the TMC proteins in mice expressing TMC1-mCherry and TMC2-AcGFP. Functionality of the tagged proteins was verified by transgenic rescue of MET currents and hearing in Tmc1(Δ/Δ);Tmc2(Δ/Δ) mice. TMC1-mCherry and TMC2-AcGFP localize along the length of immature stereocilia. However, as hair cells develop, the two proteins localize predominantly to stereocilia tips. Both TMCs are absent from the tips of the tallest stereocilia, where MET activity is not detectable. This distribution was confirmed for the endogenous proteins by immunofluorescence. These data are consistent with TMC1 and TMC2 being components of the stereocilia MET channel complex.

Integrating the biophysical and molecular mechanisms of auditory hair cell mechanotransduction

Mechanosensation is a primitive and somewhat ubiquitous sense. At the inner ear, sensory hair cells are refined to enhance sensitivity, dynamic range and frequency selectivity. Thirty years ago, mechanisms of mechanotransduction and adaptation were well accounted for by simple mechanical models that incorporated physiological and morphological properties of hair cells. Molecular and genetic tools, coupled with new optical techniques, are now identifying and localizing specific components of the mechanotransduction machinery. These new findings challenge long-standing theories, and require modification of old and development of new models. Future advances require the integration of molecular and physiological data to causally test these new hypotheses.

Gene therapy restores auditory and vestibular function in a mouse model of Usher syndrome type 1c

Because there are currently no biological treatments for hearing loss, we sought to advance gene therapy approaches to treat genetic deafness. We focused on Usher syndrome, a devastating genetic disorder that causes blindness, balance disorders and profound deafness, and studied a knock-in mouse model, Ush1c c.216G>A, for Usher syndrome type IC (USH1C). As restoration of complex auditory and balance function is likely to require gene delivery systems that target auditory and vestibular sensory cells with high efficiency, we delivered wild-type Ush1c into the inner ear of Ush1c c.216G>A mice using a synthetic adeno-associated viral vector, Anc80L65, shown to transduce 80–90% of sensory hair cells. We demonstrate recovery of gene and protein expression, restoration of sensory cell function, rescue of complex auditory function and recovery of hearing and balance behavior to near wild-type levels. The data represent unprecedented recovery of inner ear function and suggest that biological therapies to treat deafness may be suitable for translation to humans with genetic inner ear disorders.

Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents

Although genetic factors contribute to almost half of all cases of deafness, treatment options for genetic deafness are limited. We developed a genome-editing approach to target a dominantly inherited form of genetic deafness. Here we show that cationic lipid-mediated in vivo delivery of Cas9–guide RNA complexes can ameliorate hearing loss in a mouse model of human genetic deafness. We designed and validated, both in vitro and in primary fibroblasts, genome editing agents that preferentially disrupt the dominant deafness-associated allele in the Tmc1 (transmembrane channel-like gene family 1) Beethoven (Bth) mouse model, even though the mutant Tmc1Bth allele differs from the wild-type allele at only a single base pair. Injection of Cas9–guide RNA–lipid complexes targeting the Tmc1Bth allele into the cochlea of neonatal Tmc1Bth/+ mice substantially reduced progressive hearing loss. We observed higher hair cell survival rates and lower auditory brainstem response thresholds in injected ears than in uninjected ears or ears injected with control complexes that targeted an unrelated gene. Enhanced acoustic startle responses were observed among injected compared to uninjected Tmc1Bth/+ mice. These findings suggest that protein–RNA complex delivery of target gene-disrupting agents in vivo is a potential strategy for the treatment of some types of autosomal-dominant hearing loss.

A synthetic AAV vector enables safe and efficient gene transfer to the mammalian inner ear

Efforts to develop gene therapies for hearing loss have been hampered by the lack of safe, efficient, and clinically relevant delivery modalities. Here we demonstrate the safety and efficiency of Anc80L65, a rationally designed synthetic vector, for transgene delivery to the mouse cochlea. Ex vivo transduction of mouse organotypic explants identified Anc80L65 from a set of other adeno-associated virus (AAV) vectors as a potent vector for the cochlear cell targets. Round window membrane injection resulted in highly efficient transduction of inner and outer hair cells in mice, a substantial improvement over conventional AAV vectors. Anc80L65 round window injection was well tolerated, as indicated by sensory cell function, hearing and vestibular function, and immunologic parameters. The ability of Anc80L65 to target outer hair cells at high rates, a requirement for restoration of complex auditory function, may enable future gene therapies for hearing and balance disorders.

Deletion of PDZD7 disrupts the Usher syndrome type 2 protein complex in cochlear hair cells and causes hearing loss in mice

Usher syndrome type 2 (USH2) is the predominant form of USH, a leading genetic cause of combined deafness and blindness. PDZD7, a paralog of two USH causative genes, USH1C and USH2D (WHRN), was recently reported to be implicated in USH2 and non-syndromic deafness. It encodes a protein with multiple PDZ domains. To understand the biological function of PDZD7 and the pathogenic mechanism caused by PDZD7 mutations, we generated and thoroughly characterized a Pdzd7 knockout mouse model. The Pdzd7 knockout mice exhibit congenital profound deafness, as assessed by auditory brainstem response, distortion product otoacoustic emission and cochlear microphonics tests, and normal vestibular function, as assessed by their behaviors. Lack of PDZD7 leads to the disorganization of stereocilia bundles and a reduction in mechanotransduction currents and sensitivity in cochlear outer hair cells. At the molecular level, PDZD7 determines the localization of the USH2 protein complex, composed of USH2A, GPR98 and WHRN, to ankle links in developing cochlear hair cells, likely through its direct interactions with these three proteins. The localization of PDZD7 to the ankle links of cochlear hair bundles also relies on USH2 proteins. In photoreceptors of Pdzd7 knockout mice, the three USH2 proteins largely remain unchanged at the periciliary membrane complex. The electroretinogram responses of both rod and cone photoreceptors are normal in knockout mice at 1 month of age. Therefore, although the organization of the USH2 complex appears different in photoreceptors, it is clear that PDZD7 plays an essential role in organizing the USH2 complex at ankle links in developing cochlear hair cells. GenBank accession numbers: KF041446, KF041447, KF041448, KF041449, KF041450, KF041451.

TMC function in hair cell transduction

Transmembrane channel-like (TMC) proteins 1 and 2 are necessary for hair cell mechanotransduction but their precise function is controversial. A growing body of evidence supports a direct role for TMC1 and TMC2 as components of the transduction complex. However, a number of important questions remain and alternate hypotheses have been proposed. Here we present an historical overview of the identification and cloning of Tmc genes, a discussion of mutations in TMC1 that cause deafness in mice and humans and a brief review of other members of the Tmc gene superfamily. We also examine expression of Tmc mRNAs and localization of the protein products. The review focuses on potential functions of TMC proteins and the evidence from Beethoven mice that suggests a direct role for TMC1 in hair cell mechanotransduction. Data that support alternate interpretations are also considered. The article concludes with a discussion of outstanding questions and future directions for TMC research. This article is part of a Special Issue entitled .