Spontaneous allelic variant in deafness-blindness gene Ush1g resulting in an expanded phenotype.

Relationships between novel phenotypic behaviors and specific genetic alterations are often discovered using target-specific, directed mutagenesis or phenotypic selection following chemical mutagenesis. An alternative approach is to exploit deficiencies in DNA repair pathways that maintain genetic integrity in response to spontaneously induced damage. Mice deficient in the DNA glycosylase NEIL1 show elevated spontaneous mutations, which arise from translesion DNA synthesis past oxidatively induced base damage. Several litters of Neil1 knockout mice included animals that were distinguished by their backwards-walking behavior in open-field environments, while maintaining frantic forward movements in their home cage environment. Other phenotypic manifestations included swim test failures, head tilting and circling. Mapping of the mutation that conferred these behaviors showed the introduction of a stop codon at amino acid 4 of the Ush1g gene. Ush1gbw/bw null mice displayed auditory and vestibular defects that are commonly seen with mutations affecting inner-ear hair-cell function, including a complete lack of auditory brainstem responses and vestibular-evoked potentials. As in other Usher syndrome type I mutant mouse lines, hair cell phenotypes included disorganized and split hair bundles, as well as altered distribution of proteins for stereocilia that localize to the tips of row 1 or row 2. Disruption to the bundle and kinocilium displacement suggested that USH1G is essential for forming the hair cell's kinocilial links. Consistent with other Usher type 1 models, Ush1gbw/bw mice had no substantial retinal degeneration compared with Ush1gbw /+ controls. In contrast to previously described Ush1g alleles, this new allele provides the first knockout model for this gene.

GIPC3 couples to MYO6 and PDZ domain proteins, and shapes the hair cell apical region.

GIPC3 has been implicated in auditory function. Here, we establish that GIPC3 is initially localized to the cytoplasm of inner and outer hair cells of the cochlea and then is increasingly concentrated in cuticular plates and at cell junctions during postnatal development. Early postnatal Gipc3KO/KO mice had mostly normal mechanotransduction currents, but had no auditory brainstem response at 1 month of age. Cuticular plates of Gipc3KO/KO hair cells did not flatten during development as did those of controls; moreover, hair bundles were squeezed along the cochlear axis in mutant hair cells. Junctions between inner hair cells and adjacent inner phalangeal cells were also severely disrupted in Gipc3KO/KO cochleas. GIPC3 bound directly to MYO6, and the loss of MYO6 led to altered distribution of GIPC3. Immunoaffinity purification of GIPC3 from chicken inner ear extracts identified co-precipitating proteins associated with adherens junctions, intermediate filament networks and the cuticular plate. Several of immunoprecipitated proteins contained GIPC family consensus PDZ-binding motifs (PBMs), including MYO18A, which bound directly to the PDZ domain of GIPC3. We propose that GIPC3 and MYO6 couple to PBMs of cytoskeletal and cell junction proteins to shape the cuticular plate.

Control of stereocilia length during development of hair bundles.

Assembly of the hair bundle, the sensory organelle of the inner ear, depends on differential growth of actin-based stereocilia. Separate rows of stereocilia, labeled 1 through 3 from tallest to shortest, lengthen or shorten during discrete time intervals during development. We used lattice structured illumination microscopy and surface rendering to measure dimensions of stereocilia from mouse apical inner hair cells during early postnatal development; these measurements revealed a sharp transition at postnatal day 8 between stage III (row 1 and 2 widening; row 2 shortening) and stage IV (final row 1 lengthening and widening). Tip proteins that determine row 1 lengthening did not accumulate simultaneously during stages III and IV; while the actin-bundling protein EPS8 peaked at the end of stage III, GNAI3 peaked several days later-in early stage IV-and GPSM2 peaked near the end of stage IV. To establish the contributions of key macromolecular assemblies to bundle structure, we examined mouse mutants that eliminated tip links (Cdh23v2J or Pcdh15av3J), transduction channels (TmieKO), or the row 1 tip complex (Myo15ash2). Cdh23v2J/v2J and Pcdh15av3J/av3J bundles had adjacent stereocilia in the same row that were not matched in length, revealing that a major role of these cadherins is to synchronize lengths of side-by-side stereocilia. Use of the tip-link mutants also allowed us to distinguish the role of transduction from effects of transduction proteins themselves. While levels of GNAI3 and GPSM2, which stimulate stereocilia elongation, were greatly attenuated at the tips of TmieKO/KO row 1 stereocilia, they accumulated normally in Cdh23v2J/v2J and Pcdh15av3J/av3J stereocilia. These results reinforced the suggestion that the transduction proteins themselves facilitate localization of proteins in the row 1 complex. By contrast, EPS8 concentrates at tips of all TmieKO/KO, Cdh23v2J/v2J, and Pcdh15av3J/av3J stereocilia, correlating with the less polarized distribution of stereocilia lengths in these bundles. These latter results indicated that in wild-type hair cells, the transduction complex prevents accumulation of EPS8 at the tips of shorter stereocilia, causing them to shrink (rows 2 and 3) or disappear (row 4 and microvilli). Reduced rhodamine-actin labeling at row 2 stereocilia tips of tip-link and transduction mutants suggests that transduction's role is to destabilize actin filaments there. These results suggest that regulation of stereocilia length occurs through EPS8 and that CDH23 and PCDH15 regulate stereocilia lengthening beyond their role in gating mechanotransduction channels.

GIPC3 couples to MYO6 and PDZ domain proteins and shapes the hair cell apical region.

GIPC3 has been implicated in auditory function. Initially localized to the cytoplasm of inner and outer hair cells of the cochlea, GIPC3 increasingly concentrated in cuticular plates and at cell junctions during postnatal development. Early postnatal Gipc3 KO/KO mice had mostly normal mechanotransduction currents, but had no auditory brainstem response at one month of age. Cuticular plates of Gipc3 KO/KO hair cells did not flatten during development as did those of controls; moreover, hair bundles were squeezed along the cochlear axis in mutant hair cells. Junctions between inner hair cells and adjacent inner phalangeal cells were also severely disrupted in Gipc3 KO/KO cochleas. GIPC3 bound directly to MYO6, and the loss of MYO6 led to altered distribution of GIPC3. Immunoaffinity purification of GIPC3 from chicken inner ear extracts identified co-precipitating proteins associated with adherens junctions, intermediate filament networks, and the cuticular plate. Several of immunoprecipitated proteins contained GIPC-family consensus PDZ binding motifs (PBMs), including MYO18A, which binds directly to the PDZ domain of GIPC3. We propose that GIPC3 and MYO6 couple to PBMs of cytoskeletal and cell-junction proteins to shape the cuticular plate. Summary statement: The PDZ-domain protein GIPC3 couples the molecular motors MYO6 and MYO18A to actin cytoskeleton structures in hair cells. GIPC3 is necessary for shaping the hair cell’s cuticular plate and hence the arrangement of the stereocilia in the hair bundle.

ANKRD24 organizes TRIOBP to reinforce stereocilia insertion points.

The stereocilia rootlet is a key structure in vertebrate hair cells, anchoring stereocilia firmly into the cell's cuticular plate and protecting them from overstimulation. Using superresolution microscopy, we show that the ankyrin-repeat protein ANKRD24 concentrates at the stereocilia insertion point, forming a ring at the junction between the lower and upper rootlets. Annular ANKRD24 continues into the lower rootlet, where it surrounds and binds TRIOBP-5, which itself bundles rootlet F-actin. TRIOBP-5 is mislocalized in Ankrd24KO/KO hair cells, and ANKRD24 no longer localizes with rootlets in mice lacking TRIOBP-5; exogenous DsRed-TRIOBP-5 restores endogenous ANKRD24 to rootlets in these mice. Ankrd24KO/KO mice show progressive hearing loss and diminished recovery of auditory function after noise damage, as well as increased susceptibility to overstimulation of the hair bundle. We propose that ANKRD24 bridges the apical plasma membrane with the lower rootlet, maintaining a normal distribution of TRIOBP-5. Together with TRIOBP-5, ANKRD24 organizes rootlets to enable hearing with long-term resilience.

Truncation of the otoferlin transmembrane domain alters the development of hair cells and reduces membrane docking.

Release of neurotransmitter from sensory hair cells is regulated by otoferlin. Despite the importance of otoferlin in the auditory and vestibular pathways, the functional contributions of the domains of the protein have not been fully characterized. Using a zebrafish model, we investigated a mutant otoferlin with a stop codon at the start of the transmembrane domain. We found that both the phenotype severity and the expression level of mutant otoferlin changed with the age of the zebrafish. At the early developmental time point of 72 h post fertilization, low expression of the otoferlin mutant coincided with synaptic ribbon deficiencies, reduced endocytosis, and abnormal transcription of several hair cell genes. As development proceeded, expression of the mutant otoferlin increased, and both synaptic ribbons and hair cell transcript levels resembled wild type. However, hair cell endocytosis deficits and abnormalities in the expression of GABA receptors persisted even after up-regulation of mutant otoferlin. Analysis of membrane-reconstituted otoferlin measurements suggests a function for the transmembrane domain in liposome docking. We conclude that deletion of the transmembrane domain reduces membrane docking, attenuates endocytosis, and results in developmental delay of the hair cell.

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. Correspondingly, Tmc1KO/KO;Tmc2KO/KO or TmieKO/KO hair cells, which lack transduction, have significantly altered stereocilia lengths and diameters, including a narrowed row 2. EPS8 and the short splice isoform of MYO15A, identity markers for mature row 1 (the tallest row), lost their row exclusivity in transduction mutants. GNAI3, another member of the mature row 1 complex, accumulated at mutant row 1 tips at considerably lower levels than in wild-type bundles. Alterations in stereocilia dimensions and in EPS8 distribution seen in transduction mutants were mimicked by block of transduction channels of cochlear explants in culture. In addition, proteins normally concentrated at mature row 2 tips were also distributed differently in transduction mutants; the heterodimeric capping protein subunit CAPZB and its partner TWF2 never concentrated at row 2 tips like they do in wild-type bundles. The altered distribution of marker proteins in transduction mutants was accompanied by increased variability in stereocilia length. Transduction channels thus specify and maintain row identity, control addition of new actin filaments to increase stereocilia diameter, and coordinate stereocilia height within rows.

Stereocilia Rootlets: Actin-Based Structures That Are Essential for Structural Stability of the Hair Bundle.

Sensory hair cells of the inner ear rely on the hair bundle, a cluster of actin-filled stereocilia, to transduce auditory and vestibular stimuli into electrical impulses. Because they are long and thin projections, stereocilia are most prone to damage at the point where they insert into the hair cell's soma. Moreover, this is the site of stereocilia pivoting, the mechanical movement that induces transduction, which additionally weakens this area mechanically. To bolster this fragile area, hair cells construct a dense core called the rootlet at the base of each stereocilium, which extends down into the actin meshwork of the cuticular plate and firmly anchors the stereocilium. Rootlets are constructed with tightly packed actin filaments that extend from stereocilia actin filaments which are wrapped with TRIOBP; in addition, many other proteins contribute to the rootlet and its associated structures. Rootlets allow stereocilia to sustain innumerable deflections over their lifetimes and exemplify the unique manner in which sensory hair cells exploit actin and its associated proteins to carry out the function of mechanotransduction.

Otoferlin Depletion Results in Abnormal Synaptic Ribbons and Altered Intracellular Calcium Levels in Zebrafish.

The protein otoferlin plays an essential role at the sensory hair cell synapse. Mutations in otoferlin result in deafness and depending on the species, mild to strong vestibular deficits. While studies in mouse models suggest a role for otoferlin in synaptic vesicle exocytosis and endocytosis, it is unclear whether these functions are conserved across species. To address this question, we characterized the impact of otoferlin depletion in zebrafish larvae and found defects in synaptic vesicle recycling, abnormal synaptic ribbons, and higher resting calcium concentrations in hair cells. We also observed abnormal expression of the calcium binding hair cell genes s100s and parvalbumin, as well as the nogo related proteins rtn4rl2a and rtn4rl2b. Exogenous otoferlin partially restored expression of genes affected by endogenous otoferlin depletion. Our results suggest that in addition to vesicle recycling, depletion of otoferlin disrupts resting calcium levels, alters synaptic ribbon architecture, and perturbs transcription of hair cells specific genes during zebrafish development.

Otoferlin deficiency in zebrafish results in defects in balance and hearing: rescue of the balance and hearing phenotype with full-length and truncated forms of mouse otoferlin.

Sensory hair cells convert mechanical motion into chemical signals. Otoferlin, a six-C2 domain transmembrane protein linked to deafness in humans, is hypothesized to play a role in exocytosis at hair cell ribbon synapses. To date, however, otoferlin has been studied almost exclusively in mouse models, and no rescue experiments have been reported. Here we describe the phenotype associated with morpholino-induced otoferlin knockdown in zebrafish and report the results of rescue experiments conducted with full-length and truncated forms of otoferlin. We found that expression of otoferlin occurs early in development and is restricted to hair cells and the midbrain. Immunofluorescence microscopy revealed localization to both apical and basolateral regions of hair cells. Knockdown of otoferlin resulted in hearing and balance defects, as well as locomotion deficiencies. Further, otoferlin morphants had uninflated swim bladders. Rescue experiments conducted with mouse otoferlin restored hearing, balance, and inflation of the swim bladder. Remarkably, truncated forms of otoferlin retaining the C-terminal C2F domain also rescued the otoferlin knockdown phenotype, while the individual N-terminal C2A domain did not. We conclude that otoferlin plays an evolutionarily conserved role in vertebrate hearing and that truncated forms of otoferlin can rescue hearing and balance.

Nanosecond electric pulses: a novel stimulus for triggering Ca2+ influx into chromaffin cells via voltage-gated Ca2+ channels.

Exposing bovine chromaffin cells to a single 5 ns, high-voltage (5 MV/m) electric pulse stimulates Ca(2+) entry into the cells via L-type voltage-gated Ca(2+) channels (VGCC), resulting in the release of catecholamine. In this study, fluorescence imaging was used to monitor nanosecond pulse-induced effects on intracellular Ca(2+) level ([Ca(2+)](i)) to investigate the contribution of other types of VGCCs expressed in these cells in mediating Ca(2+) entry. ω-Conotoxin GVIA and ω-agatoxin IVA, antagonists of N-type and P/Q-type VGCCs, respectively, reduced the magnitude of the rise in [Ca(2+)](i) elicited by a 5 ns pulse. ω-conotoxin MVIIC, which blocks N- and P/Q-type VGCCs, had a similar effect. Blocking L-, N-, and P\Q-type channels simultaneously with a cocktail of VGCC inhibitors abolished the pulse-induced [Ca(2+)](i) response of the cells, suggesting Ca(2+) influx occurs only via VGCCs. Lowering extracellular K(+) concentration from 5 to 2 mM or pulsing cells in Na(+)-free medium suppressed the pulse-induced rise in [Ca(2+)](i) in the majority of cells. Thus, both membrane potential and Na(+) entry appear to play a role in the mechanism by which nanoelectropulses evoke Ca(2+) influx. However, activation of voltage-gated Na(+) channels (VGSC) is not involved since tetrodotoxin (TTX) failed to block the pulse-induced rise in [Ca(2+)](i). These findings demonstrate that a single electric pulse of only 5 ns duration serves as a novel stimulus to open multiple types of VGCCs in chromaffin cells in a manner involving Na(+) transport across the plasma membrane. Whether Na(+) transport occurs via non-selective cation channels and/or through lipid nanopores remains to be determined.