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Abstract Myh1 is a mouse deafness gene with an unknown function in the auditory system. Hearing loss in Myh1-knockout mice is characterized by an elevated threshold for the auditory brainstem response and the absence of a threshold for distortion product otoacoustic emission. Here, we investigated the role of MYH1 in outer hair cells (OHCs), crucial structures in the organ of Corti responsible for regulating cochlear amplification. Direct whole-cell voltage-clamp recordings of OHCs revealed that prestin activity was lower in Myh1-knockout mice than in wild-type mice, indicating abnormal OHC electromotility. We analyzed whole-exome sequencing data from 437 patients with hearing loss of unknown genetic causes and identified biallelic missense variants of MYH1 in five unrelated families. Hearing loss in individuals harboring biallelic MYH1 variants was non-progressive, with an onset ranging from congenital to childhood. Three of five individuals with MYH1 variants displayed osteopenia. Structural prediction by AlphaFold2 followed by molecular dynamic simulations revealed that the identified variants presented structural abnormalities compared with wild-type MYH1. In a heterogeneous overexpression system, MYH1 variants, particularly those in the head domain, abolished MYH1 functions, such as by increasing prestin activity and modulating the membrane traction force. Overall, our findings suggest an essential function of MYH1 in OHCs, as observed in Myh1-deficient mice, and provide genetic evidence linking biallelic MYH1 variants to autosomal recessive hearing loss in humans.

Summary: Background: Cryopyrin-associated periodic syndrome (CAPS) is an inherited autoinflammatory disease caused by a gain-of-function mutation in NLRP3. Although CAPS patients frequently suffer from sensorineural hearing loss, it remains unclear whether CAPS-associated mutation in NLRP3 is associated with the progression of hearing loss. Methods: We generated a mice with conditional expression of CAPS-associated NLRP3 mutant (D301N) in cochlea-resident CX3CR1 macrophages and examined the susceptibility of CAPS mice to inflammation-mediated hearing loss in a local and systemic inflammation context. Findings: Upon lipopolysaccharide (LPS) injection into middle ear cavity, NLRP3 mutant mice exhibited severe cochlear inflammation, inflammasome activation and hearing loss. However, this middle ear injection model induced a considerable hearing loss in control mice and inevitably caused an inflammation-independent hearing loss possibly due to ear tissue damages by injection procedure. Subsequently, we optimized a systemic LPS injection model, which induced a significant hearing loss in NLRP3 mutant mice but not in control mice. Peripheral inflammation induced by a repetitive low dose of LPS injection caused a blood-labyrinth barrier disruption, macrophage infiltration into cochlea and cochlear inflammasome activation in an NLRP3-dependent manner. Interestingly, both cochlea-infiltrating and -resident macrophages contribute to peripheral inflammation-mediated hearing loss of CAPS mice. Furthermore, NLRP3-specific inhibitor, MCC950, as well as an interleukin-1 receptor antagonist significantly alleviated systemic LPS-induced hearing loss and inflammatory phenotypes in NLRP3 mutant mice. Interpretation: Our findings reveal that CAPS-associated NLRP3 mutation is critical for peripheral inflammation-induced hearing loss in our CAPS mice model, and an NLRP3-specific inhibitor can be used to treat inflammation-mediated sensorineural hearing loss. Funding: National Research Foundation of Korea Grant funded by the Korean Government and the Team Science Award of Yonsei University College of Medicine.

Defective primary cilia cause a range of diseases known as ciliopathies, including hearing loss. The etiology of hearing loss in ciliopathies, however, remains unclear. We analyzed cochleae from three ciliopathy mouse models exhibiting different ciliogenesis defects: Intraflagellar transport 88 (Ift88), Tbc1d32 (a.k.a. bromi), and Cilk1 (a.k.a. Ick) mutants. These mutants showed multiple developmental defects including shortened cochlear duct and abnormal apical patterning of the organ of Corti. Although ciliogenic defects in cochlear hair cells such as misalignment of the kinocilium are often associated with the planar cell polarity pathway, our results showed that inner ear defects in these mutants are primarily due to loss of sonic hedgehog signaling. Furthermore, an inner ear-specific deletion of Cilk1 elicits low-frequency hearing loss attributable to cellular changes in apical cochlear identity that is dedicated to low-frequency sound detection. This type of hearing loss may account for hearing deficits in some patients with ciliopathies.

Outer hair cells (OHCs) play an essential role in hearing by acting as a nonlinear amplifier which helps the cochlea detect sounds with high sensitivity and accuracy. This nonlinear sound processing generates distortion products, which can be measured as distortion-product otoacoustic emissions (DPOAEs). The OHC stereocilia that respond to sound vibrations are connected by three kinds of extracellular links: tip links that connect the taller stereocilia to shorter ones and convey force to the mechanoelectrical transduction channels, tectorial membrane-attachment crowns (TM-ACs) that connect the tallest stereocilia to one another and to the overlying TM, and horizontal top connectors (HTCs) that link adjacent stereocilia. While the tip links have been extensively studied, the roles that the other two types of links play in hearing are much less clear, largely because of a lack of suitable animal models. Here, while analyzing genetic combinations of tubby mice, we encountered models missing both HTCs and TM-ACs or HTCs alone. We found that the tubby mutation causes loss of both HTCs and TM-ACs due to a mislocalization of stereocilin, which results in OHC dysfunction leading to severe hearing loss. Intriguingly, the addition of the modifier allele modifier of tubby hearing 1 in tubby mice selectively rescues the TM-ACs but not the HTCs. Hearing is significantly rescued in these mice with robust DPOAE production, indicating an essential role of the TM-ACs but not the HTCs in normal OHC function. In contrast, the HTCs are required for the resistance of hearing to damage caused by noise stress. [ABSTRACT FROM AUTHOR]

Sound frequency discrimination begins at the organ of Corti in mammals and the basilar papilla in birds. Both of these hearing organs are tonotopically organized such that sensory hair cells at the basal (proximal) end respond to high frequency sound, whereas their counterparts at the apex (distal) respond to low frequencies. Sonic hedgehog (Shh) secreted by the developing notochord and floor plate is required for cochlear formation in both species. In mice, the apical region of the developing cochlea, closer to the ventral midline source of Shh, requires higher levels of Shh signaling than the basal cochlea farther away from the midline. Here, gain-of-function experiments using Shh-soaked beads in ovo or a mouse model expressing constitutively activated Smoothened (transducer of Shh signaling) show up-regulation of apical genes in the basal cochlea, even though these regionally expressed genes are not necessarily conserved between the two species. In chicken, these altered gene expression patterns precede morphological and physiological changes in sensory hair cells that are typically associated with tonotopy such as the total number of stereocilia per hair cell and gene expression of an inward rectifier potassium channel, IRK1, which is a bona fide feature of apical hair cells in the basilar papilla. Furthermore, our results suggest that this conserved role of Shh in establishing cochlear tonotopy is initiated early in development by Shh emanating from the notochord and floor plate. [ABSTRACT FROM AUTHOR]