Niemann-Pick type C1 disease (NPC1) is a fatal genetic disorder caused by impaired intracellular cholesterol trafficking. Recent studies reported ototoxicity of 2-hydroxypropyl- β-cyclodextrin (HPβCD), a cholesterol chelator and the only promising treatment for NPC1. Because outer hair cells (OHCs) are the only cochlear cells affected by HPβCD, we investigated whether prestin, an OHC-specific motor protein, might be involved. Single, high-dose administration of HPβCD resulted in OHC death in prestin wildtype (WT) mice whereas OHCs were largely spared in prestin knockout (KO) mice in the basal region, implicating prestin’s involvement in ototoxicity of HPβCD. We found that prestin can interact with cholesterol in vitro, suggesting that HPβCD-induced ototoxicity may involve disruption of this interaction. Time-lapse analysis revealed that OHCs isolated from WT animals rapidly deteriorated upon HPβCD treatment while those from prestin-KOs tolerated the same regimen. These results suggest that a prestin-dependent mechanism contributes to HPβCD ototoxicity.

Synchronized beating of cilia on multiciliated cells (MCCs) generates a directional flow of mucus across epithelia. This motility requires a “9 + 2” microtubule (MT) configuration in axonemes and the unidirectional array of basal bodies of cilia on the MCCs. However, it is not fully understood what components are needed for central MT-pair assembly as they are not continuous with basal bodies in contrast to the nine outer MT doublets. In this study, we discovered that a homozygous knockdown mouse model for MT minus-end regulator calmodulin-regulated spectrin-associated protein 3 (CAMSAP3),Camsap3tm1a/tm1a, exhibited multiple phenotypes, some of which are typical of primary ciliary dyskinesia (PCD), a condition caused by motile cilia defects. Anatomical examination ofCamsap3tm1a/tm1amice revealed severe nasal airway blockage and abnormal ciliary morphologies in nasal MCCs. MCCs from different tissues exhibited defective synchronized beating and ineffective generation of directional flow likely underlying the PCD-like phenotypes. In normal mice, CAMSAP3 localized to the base of axonemes and at the basal bodies in MCCs. However, inCamsap3tm1a/tm1a, MCCs lacked CAMSAP3 at the ciliary base. Importantly, the central MT pairs were missing in the majority of cilia, and the polarity of the basal bodies was disorganized. These phenotypes were further confirmed in MCCs ofXenopusembryos when CAMSAP3 expression was knocked down by morpholino injection. Taken together, we identified CAMSAP3 as being important for the formation of central MT pairs, proper orientation of basal bodies, and synchronized beating of motile cilia.

Acquired hearing loss is the predominant neurodegenerative condition associated with aging in humans. Although mutations on several genes are known to cause congenital deafness in newborns, few genes have been implicated in age-related hearing loss (ARHL), perhaps because its cause is likely polygenic. Here, we generated mice lacking lysosomal calcium channel mucolipins 3 and 1 and discovered that both male and female mice suffered a polygenic form of hearing loss. Whereas mucolipin 1 is ubiquitously expressed in all cells, mucolipin 3 is expressed in a small subset of cochlear cells, hair cells (HCs) and marginal cells of the stria vascularis, and very few other cell types. Mice lacking both mucolipins 3 and 1, but not either one alone, experienced hearing loss as early as at 1 month of age. The severity of hearing impairment progressed from high to low frequencies and increased with age. Early onset of ARHL in these mice was accompanied by outer HC (OHC) loss. Adult mice conditionally lacking mucolipins in HCs exhibited comparable auditory phenotypes, thereby revealing that the reason for OHC loss is mucolipin codeficiency in the HCs and not in the stria vascularis. Furthermore, we observed that OHCs lacking mucolipins contained abnormally enlarged lysosomes aggregated at the apical region of the cell, whereas other organelles appeared normal. We also demonstrated that these aberrant lysosomes in OHCs lost their membrane integrity through lysosomal membrane permeabilization, a known cause of cellular toxicity that explains why and how OHCs die, leading to premature ARHL. Presbycusis, or age-related hearing loss (ARHL), is a common characteristic of aging in mammals. Although many genes have been identified to cause deafness from birth in both humans and mice, only a few are known to associate with progressive ARHL, the most prevalent form of deafness. We have found that mice lacking two lysosomal channels, mucolipins 3 and 1, suffer accelerated ARHL due to auditory outer hair cell degeneration, the most common cause of hearing loss and neurodegenerative condition in humans. Lysosomes lacking mucolipins undergo organelle membrane permeabilization and promote cytotoxicity with age, revealing a novel mechanism of outer hair cell degeneration and ARHL. These results underscore the importance of lysosomes in hair cell survival and the maintenance of hearing.

Mutations in genes encoding tectorial membrane (TM) proteins are a significant cause of human hereditary hearing loss , and several mouse models have been developed to study the functional significance of this accessory structure in the mammalian cochlea. In this study, we use otoacoustic emissions (OAE), signals obtained from the ear canal that provide a measure of cochlear function, to characterize a mouse in which the TM is detached from the spiral limbus due to an absence of otoancorin (Otoa, Lukashkin et al. 2012). Our results demonstrate that spontaneous emissions (SOAE), sounds produced in the cochlea without stimulation, increase dramatically in mice with detached TMs even though their hearing sensitivity is reduced. This behavior is unusual because wild-type (WT) controls are rarely spontaneous emitters. SOAEs in mice lacking Otoa predominate around 7 kHz, which is much lower than in either WT animals when they generate SOAEs or in mutant mice in which the TM protein Ceacam16 is absent (Cheatham et al. 2014). Although both mutants lack Hensen's stripe, loss of this TM feature is only observed in regions coding frequencies greater than~15 kHz in WT mice so its loss cannot explain the low-frequency, de novo SOAEs observed in mice lacking Otoa. The fact that~80 % of mice lacking Otoa produce SOAEs even when they generate smaller distortion product OAEs suggests that the active process is still functioning in these mutants but the system(s) involved have become less stable due to alterations in TM structure.