Fetal antisense oligonucleotide therapy for congenital deafness and vestibular dysfunction.

Disabling hearing loss impacts ∼466 million individuals worldwide with 34 million children affected. Gene and pharmacotherapeutic strategies to rescue auditory function in mouse models of human deafness are most effective when administered before hearing onset, after which therapeutic efficacy is significantly diminished or lost. We hypothesize that preemptive correction of a mutation in the fetal inner ear prior to maturation of the sensory epithelium will optimally restore sensory function.

Mechanotransduction-Dependent Control of Stereocilia Dimensions and Row Identity in Inner Hair Cells.

Actin-rich structures, like stereocilia and microvilli, are assembled with precise control of length, diameter, and relative spacing. By quantifying actin-core dimensions of stereocilia from phalloidin-labeled mouse cochleas, we demonstrated that inner hair cell stereocilia developed in specific stages, where a widening phase is sandwiched between two lengthening phases. Moreover, widening of the second-tallest stereocilia rank (row 2) occurred simultaneously with the appearance of mechanotransduction.

Electron cryo-tomography of vestibular hair-cell stereocilia.

High-resolution imaging of hair-cell stereocilia of the inner ear has contributed substantially to our understanding of auditory and vestibular function. To provide three-dimensional views of the structure of stereocilia cytoskeleton and membranes, we developed a method for rapidly freezing unfixed stereocilia on electron microscopy grids, which allowed subsequent 3D imaging by electron cryo-tomography. Structures of stereocilia tips, shafts, and tapers were revealed, demonstrating that the actin paracrystal was not perfectly ordered.

Transcriptional Dynamics of Hair-Bundle Morphogenesis Revealed with CellTrails.

Protruding from the apical surface of inner ear sensory cells, hair bundles carry out mechanotransduction. Bundle growth involves sequential and overlapping cellular processes, which are concealed within gene expression profiles of individual cells. To dissect such processes, we developed CellTrails, a tool for uncovering, analyzing, and visualizing single-cell gene-expression dynamics.

ELMOD1 Stimulates ARF6-GTP Hydrolysis to Stabilize Apical Structures in Developing Vestibular Hair Cells.

Sensory hair cells require control of physical properties of their apical plasma membranes for normal development and function. Members of the ADP-ribosylation factor (ARF) small GTPase family regulate membrane trafficking and cytoskeletal assembly in many cells. We identified ELMO domain-containing protein 1 (ELMOD1), a guanine nucleoside triphosphatase activating protein (GAP) for ARF6, as the most highly enriched ARF regulator in hair cells.

Heterodimeric capping protein is required for stereocilia length and width regulation.

Control of the dimensions of actin-rich processes like filopodia, lamellipodia, microvilli, and stereocilia requires the coordinated activity of many proteins. Each of these actin structures relies on heterodimeric capping protein (CAPZ), which blocks actin polymerization at barbed ends. Because dimension control of the inner ear's stereocilia is particularly precise, we studied the CAPZB subunit in hair cells.

Plastin 1 widens stereocilia by transforming actin filament packing from hexagonal to liquid.

With their essential role in inner ear function, stereocilia of sensory hair cells demonstrate the importance of cellular actin protrusions. Actin packing in stereocilia is mediated by cross-linkers of the plastin, fascin, and espin families. Although mice lacking espin (ESPN) have no vestibular or auditory function, we found that mice that either lacked plastin 1 (PLS1) or had nonfunctional fascin 2 (FSCN2) had reduced inner ear function, with double-mutant mice most strongly affected.

The R109H variant of fascin-2, a developmentally regulated actin crosslinker in hair-cell stereocilia, underlies early-onset hearing loss of DBA/2J mice.

The quantitative trait locus ahl8 is a key contributor to the early-onset, age-related hearing loss of DBA/2J mice. A nonsynonymous nucleotide substitution in the mouse fascin-2 gene (Fscn2) is responsible for this phenotype, confirmed by wild-type BAC transgene rescue of hearing loss in DBA/2J mice. In chickens and mice, FSCN2 protein is abundant in hair-cell stereocilia, the actin-rich structures comprising the mechanically sensitive hair bundle, and is concentrated toward stereocilia tips of the bundle's longest stereocilia.

Harmonin mutations cause mechanotransduction defects in cochlear hair cells.

In hair cells, mechanotransduction channels are gated by tip links, the extracellular filaments that consist of cadherin 23 (CDH23) and protocadherin 15 (PCDH15) and connect the stereocilia of each hair cell. However, which molecules mediate cadherin function at tip links is not known. Here we show that the PDZ-domain protein harmonin is a component of the upper tip-link density (UTLD), where CDH23 inserts into the stereociliary membrane.

Splice-site A choice targets plasma-membrane Ca2+-ATPase isoform 2 to hair bundles.

Localization of mechanotransduction in sensory hair cells to hair bundles requires selective targeting of essential proteins to specific locations. Isoform 2 of the plasma-membrane Ca2+-ATPase (PMCA2), required for hearing and balance, is found exclusively in hair bundles. We determined the contribution of splicing at the two major splicing sites (A and C) to hair-cell targeting of PMCA2.

Have we found the tip link, transduction channel, and gating spring of the hair cell?

Recent reports have offered candidates for key components of the apparatus used for mechanotransduction in hair cells. TRPA1 and cadherin 23 have been proposed to be the transduction channel and component of the tip link, respectively; moreover, ankyrin repeats in TRPA1 have been proposed to be the gating spring. Although these are excellent candidates for the three components, definitive experiments supporting each identification have yet to be performed.

Cadherin 23 is a component of the tip link in hair-cell stereocilia.

Mechanoelectrical transduction, the conversion of mechanical force into electrochemical signals, underlies a range of sensory phenomena, including touch, hearing and balance. Hair cells of the vertebrate inner ear are specialized mechanosensors that transduce mechanical forces arising from sound waves and head movement to provide our senses of hearing and balance; however, the mechanotransduction channel of hair cells and the molecules that regulate channel activity have remained elusive. One molecule that might participate in mechanoelectrical transduction is cadherin 23 (CDH23), as mutations in its gene cause deafness and age-related hearing loss.

Myosin-I isozymes in neonatal rodent auditory and vestibular epithelia.

Myosin isozymes are essential for hair cells, the sensory cells of the inner ear. Because a myosin-I subfamily member may mediate adaptation of mechanoelectrical transduction, we examined expression of all eight myosin-I isozymes in rodent auditory and vestibular epithelia. Using RT-PCR, we found prominent expression of three isozymes, Myo1b (also known as myosin-Ia or myr 1).

Myosin-1c interacts with hair-cell receptors through its calmodulin-binding IQ domains.

Myosin-1c plays an essential role in adaptation of hair-cell mechanoelectrical transduction. To mediate adaptation, myosin-1c must interact directly or indirectly with other components of the transduction apparatus, including the mechanically gated transduction channel. As a first step toward identifying myosin-1c receptors, we used recombinant myosin-1c fragments to identify specific binding sites in hair cells and to biochemically characterize their interaction with myosin-1c.