Jing Zheng

Oncomodulin (OCM) is the most abundant Ca²⁺ buffering protein found in mature outer hair cells (OHCs). Cadherin 23 (CDH23) is a crucial component of the tip-links in hair cell stereocilia. The absence or dysfunction of these two proteins contributes to the early onset of age-related hearing loss (AHL). In this study, we investigated the effects of the Cdh23^753G→A mutation on OHC function using new Ocm-knockout (KO) mouse models (Ocm^tm1a/tm1a) with or without the Cdh23^753G→A mutation. Despite having the same genetic background, Ocm-KO mice carrying the Cdh23^753G→A mutation displayed a notable decline in OHC function across all measured frequencies as early as three months of age. In contrast, Ocm-KO mice without the Cdh23^753G→A mutation did not exhibit comparable hearing loss until they reached twelve months of age. Additionally, we examined the role of OCM in preserving OHC function under ototoxic stress induced by HPβCD (2-hydroxypropyl-β-cyclodextrin). The distortion product otoacoustic emission data show that the administration of HPβCD resulted in a more pronounced decline in OHC function in Ocm-KO mice compared to wild-type (WT) mice. Time-lapse recording also shows that HPβCD treatment led to greater structural deterioration and more rapid rupture events in OHCs from Ocm-KO mice than in those from WT mice. These findings suggest that the Cdh23^753G→A mutation, rather than other potential strain-specific genetic factors associated with AHL, significantly exacerbates the early onset of AHL phenotypes in Ocm-KO mice. Furthermore, our data indicates that the OCM protein in OHCs enhances their ability to withstand ototoxic stimuli.

Oncomodulin (OCM) is the most abundant Ca2+ buffering protein found in mature outer hair cells (OHCs). Cadherin 23 (CDH23) is a crucial component of the tip-links in hair cell stereocilia. The absence or dysfunction of these two proteins contributes to the early onset of age-related hearing loss (AHL). In this study, we investigated the effects of the Cdh23^753G→A mutation on OHC function using new Ocm-knockout (KO) mouse models (Ocm^tm1a/tm1a) with or without the Cdh23^753G→A mutation. Despite having the same genetic background, Ocm-KO mice carrying the Cdh23^753G→A mutation displayed significant hearing loss and a notable decline in OHC function across all measured frequencies as early as three months of age. In contrast, Ocm-KO mice without the Cdh23^753G→A mutation did not exhibit comparable hearing loss until they reached twelve months of age. Additionally, we examined the role of OCM in preserving OHC function under ototoxic stress induced by HPβCD (2-hydroxypropyl-β-cyclodextrin). The administration of HPβCD resulted in a more pronounced decline in OHC function in Ocm-KO mice compared to wild-type (WT) mice, as indicated by distortion product otoacoustic emissions (DPOAE) measurements. Furthermore, HPβCD treatment led to greater structural deterioration and more rapid rupture events in OHCs from Ocm-KO mice than in those from WT mice, as demonstrated by in vitro live imaging. These findings suggest that the Cdh23^753G→A mutation, rather than other potential strain-specific genetic factors associated with AHL, solely exacerbates the early onset of AHL phenotypes in Ocm-KO mice. Furthermore, our data indicates that OCM proteins in OHCs enhance their ability to withstand ototoxic stimuli.

The motor protein prestin, found in the inner ear’s outer hair cells (OHCs), is responsible for high sensitivity and sharp frequency selectivity in mammalian hearing. Some studies have suggested that prestin could be a serological biomarker for cochlear damage, as OHCs are highly vulnerable to damage from various sources. However, the reported data are inconsistent and lack appropriate negative controls. To investigate whether prestin can be used as a serological biomarker for cochlear damage or stress, we measured prestin quantities in the bloodstreams of mice using ELISA kits from different companies. Wildtype (WT) mice were exposed to different ototoxic treatments, including noise exposure and ototoxic reagents that rapidly kill OHCs. Prestin-knockout (KO) mice were used as a negative control. Our data show that some ELISA kits were not able to detect prestin specifically. The ELISA kit that could detect the prestin protein from cochlear homogenates failed to detect prestin in the bloodstream, despite there being significant damage to OHCs in the cochleae. Furthermore, the optical densities of the serum samples, which correlate to prestin quantities, were significantly influenced by hemolysis in the samples. In conclusion, Prestin from OHCs is not a sensitive and reliable serological biomarker for detecting cochlear damage in mice using ELISA.

The motor protein Prestin found in the outer hair cells (OHCs) of the inner ear is responsible for producing high sensitivity and sharp frequency selectivity for mammalian hearing. Some studies have suggested that Prestin could serve as a serological biomarker for cochlear damage since OHCs are highly vulnerable to damage from various sources of insults. However, the reported data are inconsistent and lack appropriate negative controls. To investigate this further, we measured Prestin quantities in the bloodstream using ELISA kits from different companies. Wildtype (WT) mice were exposed to different ototoxic treatments, including noise exposure and ototoxic reagents that kill OHCs rapidly. Prestin-knockout (KO) mice were used as a negative control. Our data show that some ELISA kits were not able to detect Prestin specifically. The ELISA kit that could detect Prestin protein from cochlear homogenates failed to detect Prestin in the bloodstream despite significant damage to OHCs in cochleae. Furthermore, the optical densities of samples, which correlate to Prestin quantities, were significantly influenced by hemolysis in the samples. In conclusion, Prestin from OHCs is not a sensitive and reliable serological biomarker for detecting cochlear damage in mice.

Kinocilia are exceptionally long primary sensory cilia located on vestibular hair cells, which are essential for transmitting key signals that contribute to mammalian balance and overall vestibular system function. Kinocilia have a “9+2” microtubule (MT) configuration with nine doublet MTs surrounding two central singlet MTs. This is uncommon as most mammalian primary sensory cilia have a “9+0” configuration, in which the central MT pair is absent. It has yet to be determined what the function of the central MT pair is in kinocilia. Calmodulin-regulated spectrin-associated protein 3 (CAMSAP3) regulates the minus end of MTs and is essential for forming the central MT pair in motile cilia, which have the “9+2” configuration. To explore the role of the central MT pair in kinocilia, we created a conditional knockout model (cKO), Camsap3-cKO, which intended to eliminate CAMSAP3 in limited organs including the inner ear, olfactory bulb, and kidneys. Immunofluorescent staining of vestibular organs demonstrated that CAMSAP3 proteins were significantly reduced in Camsap3-cKO mice and that aged Camsap3-cKO mice had significantly shorter kinocilia than their wildtype littermates. Transmission electron microscopy showed that aged Camsap3-cKO mice were in fact missing that the central MT pair in kinocilia more often than their wildtype counterparts. In the examination of behavior, wildtype and Camsap3-cKO mice performed equally well on a swim assessment, right-reflex test, and evaluation of balance on a rotarod. However, Camsap3-cKO mice showed slightly altered gaits including reduced maximal rate of change of paw area and a smaller paw area in contact with the surface. Although Camsap3-cKO mice had no differences in olfaction from their wildtype counterparts, Camsap3-cKO mice did have kidney dysfunction that deteriorated their health. Thus, CAMSAP3 is important for establishing and/or maintaining the normal structure of kinocilia and kidney function but is not essential for normal olfaction. Our data supports our hypothesis that CAMSAP3 is critical for construction of the central MT pair in kinocilia, and that the central MT pair may be important for building long and stable axonemes in these kinocilia. Whether shorter kinocilia might lead to abnormal vestibular function and altered gaits in older Camsap3-cKO mice requires further investigation.

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), Camsap3^tm1a/tm1a, exhibited multiple phenotypes, some of which are typical of primary ciliary dyskinesia (PCD), a condition caused by motile cilia defects. Anatomical examination of Camsap3^tm1a/tm1a mice 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, in Camsap3^tm1a/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 of Xenopus embryos 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. Significance Cilia are composed of hundreds of proteins whose identities and functions are far from being completely understood. In this study, we determined that calmodulin-regulated spectrin-associated protein 3 (CAMSAP3) plays an important role for the function of motile cilia in multiciliated cells (MCCs). Global knockdown of CAMSAP3 protein expression in mice resulted in defects in ciliary structures, polarity, and synchronized beating in MCCs. These animals also displayed signs and symptoms reminiscent of primary ciliary dyskinesia (PCD), including a mild form of hydrocephalus, subfertility, and impaired mucociliary clearance that leads to hyposmia, anosmia, rhinosinusitis, and otitis media. Functional characterization of CAMSAP3 enriches our understanding of the molecular mechanisms underlying the generation and function of motile cilia in MCCs.

Niemann-Pick Type C1 (NPC1) disease is a fatal neurovisceral disorder caused by dysfunction of NPC1 protein, which plays a role in intracellular cholesterol trafficking. The cholesterol-chelating agent, 2-hydroxypropyl-β-cyclodextrin (HPβCD), is currently undergoing clinical trials for treatment of this disease. Though promising in alleviating neurological symptoms, HPβCD causes irreversible hearing loss in NPC1 patients and outer hair cell (OHC) death in animal models. We recently found that HPβCD-induced OHC death can be significantly alleviated in a mouse model lacking prestin, an OHC-specific motor protein required for the high sensitivity and sharp frequency selectivity of mammalian hearing. Since cholesterol status is known to influence prestin’s electromotility, we examined how prestin contributes to HPβCD-induced OHC death in the disease context using the NPC1 knockout (KO) mouse model (NPC1-KO). We found normal expression and localization of prestin in NPC1-KO OHCs. Whole-cell patch-clamp recordings revealed a significant depolarization of the voltage-operating point of prestin in NPC1-KO mice, suggesting reduced levels of cholesterol in the lateral membrane of OHCs that lack NPC1. OHC loss and elevated thresholds were found for high frequency regions in NPC1-KO mice, whose OHCs retained their sensitivity to HPβCD. To investigate whether prestin’s electromotile function contributes to HPβCD-induced OHC death, the prestin inhibitor salicylate was co-administered with HPβCD to WT and NPC1-KO mice. Neither oral nor intraperitoneal administration of salicylate mitigated HPβCD-induced OHC loss. To further determine the contribution of prestin’s electromotile function, a mouse model expressing a virtually nonelectromotile prestin protein (499-prestin) was subjected to HPβCD treatment. 499-prestin knockin mice showed no resistance to HPβCD-induced OHC loss. As 499-prestin maintains its ability to bind cholesterol, our data imply that HPβCD-induced OHC death is ascribed to the structural role of prestin in maintaining the OHC’s lateral membrane, rather than its motor function.

Outer hair cells (OHC) act as amplifiers and their function is modified by medial olivocochlear (MOC) efferents. The unique OHC motor protein, prestin, provides the molecular basis for somatic electromotility, which is required for sensitivity and frequency selectivity, the hallmarks of mammalian hearing. Prestin proteins are the major component of the lateral membrane of mature OHCs, which separates apical and basal domains. To investigate the contribution of prestin to this unique arrangement, we compared the distribution of membrane proteins in OHCs of wildtype (WT) and prestin-knockout (KO) mice. In WT, the apical protein PMCA2 was exclusively localized to the hair bundles, while it was also found at the lateral membrane in KOs. Similarly, a basal protein KCNQ4 did not coalesce at the base of OHCs but was widely dispersed in mice lacking prestin. Since the expression levels of PMCA2 and KCNQ4 remained unchanged in KOs, the data indicate that prestin is required for the normal distribution of apical and basal membrane proteins in OHCs. Since OHC synapses predominate in the basal subnuclear region, we also examined the synaptic architecture in prestin-KO mice. Although neurite densities were not affected, MOC efferent terminals in prestin-KO mice were no longer constrained to the basal pole as in WT. This trend was evident as early as at postnatal day 12. Furthermore, terminals were often enlarged and frequently appeared as singlets when compared to the multiple clusters of individual terminals in WT. This abnormality in MOC synaptic morphology in prestin-KO mice is similar to defects in mice lacking MOC pathway proteins such as α9/α10 nicotinic acetylcholine receptors and BK channels, indicating a role for prestin in the proper establishment of MOC synapses. To investigate the contribution of prestin’s electromotility, we also examined OHCs from a mouse model that expresses non-functional prestin (499-prestin). We found no changes in PMCA2 localization and MOC synaptic morphology in OHCs from 499-prestin mice. Taken together, these results indicate that prestin, independent of its motile function, plays an important structural role in membrane compartmentalization, which is required for the formation of normal efferent-OHC synapses in mature OHCs.

Cadherin-related 23 (CDH23) is an adhesive protein important for hearing and vision, while CAMSAP3/Marshalin is a microtubule (MT) minus-end binding protein that regulates MT networks. Although both CDH23 and CAMSAP3/Marshalin are expressed in the organ of Corti and carry several protein-protein interaction domains, no functional connection between these two proteins has been proposed. In this report, we demonstrate that the C isoform of CDH23 (CDH23-C) directly binds to CAMSAP3/Marshalin and modifies its function by inhibiting CAMSAP3/Marshalin-induced bundle formation, a process that requires a tubulin-binding domain called CKK. We further identified a conserved N-terminal region of CDH23-C that binds to the CKK domain. This CKK binding motif (CBM) is adjacent to the domain that interacts with harmonin, a binding partner of CDH23 implicated in deafness. Because the human Usher Syndrome 1D-associated mutation, CDH23 R3175H, maps to the CBM, we created a matched mutation in mouse CDH23-C at R55H. Both in vivo and in vitro assays decreased the ability of CDH23-C to interact with CAMSAP3/Marshalin, indicating that the interaction between CDH23 and CAMSAP3/Marshalin plays a vital role in hearing and vision. Together, our data suggest that CDH23-C is a CAMSAP3/Marshalin-binding protein that can modify MT networks indirectly through its interaction with CAMSAP3/Marshalin.