Disruption of Hars2 in Cochlear Hair Cells Causes Progressive Mitochondrial Dysfunction and Hearing Loss in Mice

Mutations in a number of genes encoding mitochondrial aminoacyl-tRNA synthetases lead to non-syndromic and/or syndromic sensorineural hearing loss in humans, while their cellular and physiological pathology in cochlea has rarely been investigated in vivo. In this study, we showed that histidyl-tRNA synthetase HARS2, whose deficiency is associated with Perrault syndrome 2 (PRLTS2), is robustly expressed in postnatal mouse cochlea including the outer and inner hair cells. Targeted knockout of Hars2 in mouse hair cells resulted in delayed onset (P30), rapidly progressive hearing loss similar to the PRLTS2 hearing phenotype. Significant hair cell loss was observed starting from P45 following elevated reactive oxygen species (ROS) level and activated mitochondrial apoptotic pathway. Despite of normal ribbon synapse formation, whole-cell patch clamp of the inner hair cells revealed reduced calcium influx and compromised sustained synaptic exocytosis prior to the hair cell loss at P30, consistent with the decreased supra-threshold wave I amplitudes of the auditory brainstem response. Starting from P14, increasing proportion of morphologically abnormal mitochondria was observed by transmission electron microscope, exhibiting swelling, deformation, loss of cristae and emergence of large intrinsic vacuoles that are associated with mitochondrial dysfunction. Though the mitochondrial abnormalities are more prominent in inner hair cells, it is the outer hair cells suffering more severe cell loss. Taken together, our results suggest that conditional knockout of Hars2 in mouse cochlear hair cells leads to accumulating mitochondrial dysfunction and ROS stress, triggers progressive hearing loss highlighted by hair cell synaptopathy and apoptosis, and is differentially perceived by inner and outer hair cells.

SMPX Deficiency Causes Stereocilia Degeneration and Progressive Hearing Loss in CBA/CaJ Mice

The small muscle protein, x-linked (SMPX) encodes a small protein containing 88 amino acids. Malfunction of this protein can cause a sex-linked non-syndromic hearing loss, named X-linked deafness 4 (DFNX4). Herein, we reported a point mutation and a frameshift mutation in two Chinese families who developed gradual hearing loss with age. To explore the impaired sites in the hearing system and the mechanism of DFNX4, we established and validated an Smpx null mouse model using CRISPR-Cas9. By analyzing auditory brainstem response (ABR), male Smpx null mice showed a progressive hearing loss starting from high frequency at the 3rd month. Hearing loss in female mice was milder and occurred later compared to male mice, which was very similar to human beings. Through morphological analyses of mice cochleas, we found the hair cell bundles progressively degenerated from the shortest row. Cellular edema occurred at the end phase of stereocilia degeneration, followed by cell death. By transfecting exogenous fluorescent Smpx into living hair cells, Smpx was observed to be expressed in stereocilia. Through noise exposure, it was shown that Smpx might participate in maintaining hair cell bundles. This Smpx knock-out mouse might be used as a suitable model to explore the pathology of DFNX4.

Audiological Patterns in Patients with Autoimmune Hearing Loss

<b><i>Introduction:</i></b> The aim of this study was to illustrate clinical and audiological patterns of hearing impairment in patients with autoimmune hearing loss (AIHL). <b><i>Methods:</i></b> Fifty-three patients with AIHL were retrospectively recruited, and a tapering schema of steroid treatment was administered in all these patients. The diagnosis of AIHL was essentially based on clinical symptoms, such as recurrent, sudden (sensorineural hearing loss [SSHL]), fluctuating, or quickly progressing (&#x3c;12 months) SSHL (uni-/bilateral), in association with the coexistence of autoimmune diseases, high antinuclear antibodies (ANA) and the presence of human leukocyte antigen (HLA) B27, B35, B51, C04, and C07. Logistic regression analysis was applied to correlate the clinical data and laboratory features of AIHL with final outcomes. <b><i>Results:</i></b> The onset of AIHL was mainly progressive (49%), followed by SSHL (39.6%) or fluctuating (11.3%). The pure-tone audiogram showed more commonly a downsloping pattern (42.6% of ears), but also an upsloping, flat, cookie-bite, or inverse cookie-bite shape. Bilateral progressive AIHL was more frequently simultaneous (23 patients) than heterochronous (4 patients). Nineteen patients (35.8%) showed a favorable response to steroid therapy. The presence of recurrent, bilateral SSHL versus recurrent, unilateral SSHL had statistically negative effect on hearing recovery (OR = 0.042, <i>p</i> &#x3c; 0.05). The heterochronous bilateral SSHL may have better prognosis than simultaneous bilateral SSHL (OR = 10.000, <i>p</i> = 0.099). The gender, age, concomitant autoimmune disease, high ANA, HLA alleles, tinnitus, and vestibular symptoms had no statistical effect on a favorable outcome of AIHL. <b><i>Conclusions:</i></b> A bilateral, simultaneous, and progressive hearing loss combined with downsloping audiogram occurred more often in patients with AIHL. Bilateral simultaneous SSHL with recurrences represents the worse prognostic form of AIHL.

Myosin-driven Nucleation of Actin Filaments Drives Stereocilia Development Critical for Hearing

The assembly and maintenance of actin-based mechanosensitive stereocilia in the cochlea is critical for lifelong hearing. Myosin-15 (MYO15) is hypothesized to modulate stereocilia height by trafficking actin regulatory proteins to their tip compartments, where actin polymerization must be precisely controlled during development. We identified a mutation (p.D1647G) in the MYO15 motor-domain that initially maintained trafficking, but caused progressive hearing loss by stunting stereocilia growth, revealing an additional function for MYO15. Consistent with its maintenance of tip trafficking in vivo, purified p.D1647G MYO15 modestly reduced actin-stimulated ATPase activity in vitro. Using ensemble and single-filament fluorescence in vitro assays, we demonstrated that wild-type MYO15 directly accelerated actin filament polymerization by driving nucleation, whilst p.D1647G MYO15 blocked this activity. Collectively, our studies suggest direct actin nucleation by MYO15 at the stereocilia tip is necessary for elongation in vivo, and that this is a primary mechanism disrupted in DFNB3 hereditary human hearing loss.

Structural basis for tunable control of actin dynamics by myosin-15 in mechanosensory stereocilia

The motor protein myosin-15 is necessary for the development and maintenance of mechanosensory stereocilia, and myosin-15 mutations cause profound deafness. In a companion study, we report that myosin-15 nucleates actin filament ("F-actin") assembly and identify a progressive hearing loss mutation (p.D1647G, "jordan") which disrupts stereocilia elongation by inhibiting actin polymerization. Here, we present cryo-EM structures of myosin-15 bound to F-actin, providing a framework for interpreting deafness mutations and their impacts on myosin-stimulated actin assembly. Rigor myosin-15 evokes conformational changes in F-actin yet maintains flexibility in actin's D-loop, which mediates inter-subunit contacts, while the jordan mutant locks the D-loop in a single conformation. ADP-bound myosin-15 also locks the D-loop, which correspondingly blunts actin-polymerization stimulation. We propose myosin-15 enhances polymerization by bridging actin protomers, regulating nucleation efficiency by modulating actin's structural plasticity in a myosin nucleotide-state dependent manner. This tunable regulation of actin polymerization could be harnessed to precisely control stereocilium height.

Oncomodulin Delays Age-Related Hearing Loss in C57 and CBA Mice

Ca2+ signaling is a major contributor to sensory hair cell function in the cochlea. Oncomodulin (OCM) is a Ca2+ binding protein preferentially expressed in outer hair cells of the cochlea and few other specialized cell types. Here, we expand on our previous reports and show that OCM prevents early progressive hearing loss in mice of two different genetic backgrounds: CBA/CaJ and C57Bl/6J. In both backgrounds, genetic disruption of Ocm leads to early progressive hearing loss as measured by auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE). In both strains, loss of Ocm reduced hearing across lifetime (hearing span) by more than 50% relative to wild type (WT). Even though the two WT strains have very different hearing spans, OCM plays a considerable and similar role within their genetic environment to regulate hearing function. The accelerated ARHL of the Ocm KO illustrates the importance of Ca2+ signaling in maintaining hearing health.

Targeted Deletion of Loxl3 by Col2a1-Cre Leads to Progressive Hearing Loss

Collagens are major constituents of the extracellular matrix (ECM) that play an essential role in the structure of the inner ear and provide elasticity and rigidity when the signals of sound are received and transformed into electrical signals. LOXL3 is a member of the lysyl oxidase (LOX) family that are copper-dependent amine oxidases, generating covalent cross-links to stabilize polymeric elastin and collagen fibers in the ECM. Biallelic missense variant of LOXL3 was found in Stickler syndrome with mild conductive hearing loss. However, available information regarding the specific roles of LOXL3 in auditory function is limited. In this study, we showed that the Col2a1-Cre-mediated ablation of Loxl3 in the inner ear can cause progressive hearing loss, degeneration of hair cells and secondary degeneration of spiral ganglion neurons. The abnormal distribution of type II collagen in the spiral ligament and increased inflammatory responses were also found in Col2a1–Loxl3–/– mice. Amino oxidase activity exerts an effect on collagen; thus, Loxl3 deficiency was expected to result in the instability of collagen in the spiral ligament and the basilar membrane, which may interfere with the mechanical properties of the organ of Corti and induce the inflammatory responses that are responsible for the hearing loss. Overall, our findings suggest that Loxl3 may play an essential role in maintaining hearing function.