Molecular Remodeling of Tip Links Underlies Mechanosensory Regeneration in Auditory Hair Cells

Sound detection by inner ear hair cells requires tip links that interconnect mechanosensory stereocilia and convey force to yet unidentified transduction channels. Current models postulate a static composition of the tip link, with protocadherin 15 (PCDH15) at the lower and cadherin 23 (CDH23) at the upper end of the link. In terminally differentiated mammalian auditory hair cells, tip links are subjected to sound-induced forces throughout an organism\u27s life. Although hair cells can regenerate disrupted tip links and restore hearing, the molecular details of this process are unknown. We developed a novel implementation of backscatter electron scanning microscopy to visualize simultaneously immuno-gold particles and stereocilia links, both of only a few nanometers in diameter. We show that functional, mechanotransduction-mediating tip links have at least two molecular compositions, containing either PCDH15/CDH23 or PCDH15/PCDH15. During regeneration, shorter tip links containing nearly equal amounts of PCDH15 at both ends appear first. Whole-cell patch-clamp recordings demonstrate that these transient PCDH15/PCDH15 links mediate mechanotransduction currents of normal amplitude but abnormal Ca(2+)-dependent decay (adaptation). The mature PCDH15/CDH23 tip link composition is re-established later, concomitant with complete recovery of adaptation. Thus, our findings provide a molecular mechanism for regeneration and maintenance of mechanosensory function in postmitotic auditory hair cells and could help identify elusive components of the mechanotransduction machinery

Nanoscale live-cell imaging using hopping probe ion conductance microscopy,

We describe hopping mode scanning ion conductance microscopy that allows noncontact imaging of the complex three-dimensional surfaces of live cells with resolution better than 20 nm. We tested the effectiveness of this technique by imaging networks of cultured rat hippocampal neurons and mechanosensory stereocilia of mouse cochlear hair cells. The technique allowed examination of nanoscale phenomena on the surface of live cells under physiological conditions. There is a great interest in developing methods to image live cells at nanoscale resolution. Scanning probe microscopy (SPM) is one approach to this problem and both atomic force microscopy (AFM) and scanning electrochemical microscopy (SECM) have been used to image live cells 1,2 . However, deformation of the soft and responsive cell by the AFM cantilever, particularly when imaging eukaryotic cells, represents a substantial problem for AFM. SECM, in contrast, involves no physical contact with the sample, but true topographic imaging of the convoluted surface of living cells with nanoscale resolution has not been reported. Scanning ion conductance microscopy (SICM) 3 is another form of SPM, which allows imaging of the cell surface under physiological conditions without physical contact and with a resolution of 3-6 nm 4,5 . Until now, SICM has been restricted to imaging relatively flat surfaces, as all other SPM techniques. This is because when the probe encounters a vertical structure, it inevitably collides with the specimen SICM is based on the phenomenon that the ion flow through a sharp fluid-filled nanopipette is partially occluded when the pipette approaches the surface of a cell 3 . In conventional SICM, a nanopipette is mounted on a three-dimensional piezoelectric translation stage and automatic feedback control moves the pipette up or down to keep the pipette current constant (the set point) while the sample is scanned in x and y directions. Thus, a pipette-sample separation, typically equal to the pipette's inner radius, is maintained during imaging. In hopping probe ion conductance microscopy (HPICM), we no longer use continuous feedback. Instead, at each imaging point, the pipette approaches the sample from a starting position that is above any of the surface features We illustrate the benefits of HPICM in In contrast to conventional raster scanning, HPICM has the additional advantage that the order of imaging pixels is not predetermined. Therefore, we divided the entire image into equal-sized square

Noise exposure immediately activates cochlear mitogen-activated protein kinase signaling

Noise-induced hearing loss (NIHL) is a major public health issue worldwide. Uncovering the early molecular events associated with NIHL would reveal mechanisms leading to the hearing loss. Our aim is to investigate the immediate molecular responses after different levels of noise exposure and identify the common and distinct pathways that mediate NIHL. Previous work showed mice exposed to 116 decibels sound pressure level (dB SPL) broadband noise for 1 h had greater threshold shifts than the mice exposed to 110 dB SPL broadband noise, hence we used these two noise levels in this study. Groups of 4-8-week-old CBA/CaJ mice were exposed to no noise (control) or to broadband noise for 1 h, followed by transcriptome analysis of total cochlear RNA isolated immediately after noise exposure. Previously identified and novel genes were found in all data sets. Following exposure to noise at 116 dB SPL, the earliest responses included up-regulation of 243 genes and down-regulation of 61 genes, while a similar exposure at 110 dB SPL up-regulated 155 genes and down-regulated 221 genes. Bioinformatics analysis indicated that mitogen-activated protein kinase (MAPK) signaling was the major pathway in both levels of noise exposure. Nevertheless, both qualitative and quantitative differences were noticed in some MAPK signaling genes, after exposure to different noise levels. Cacna1b , Cacna1g , and Pla2g6 , related to calcium signaling were down-regulated after 110 dB SPL exposure, while the fold increase in the expression of Fos was relatively lower than what was observed after 116 dB SPL exposure. These subtle variations provide insight on the factors that may contribute to the differences in NIHL despite the activation of a common pathway

Inhibition of Mitochondrial Division Attenuates Cisplatin-Induced Toxicity in the Neuromast Hair Cells

Cisplatin and other related platinum antineoplastic drugs are commonly used in the treatment of a variety of cancers in both adults and children but are often associated with severe side effects, including hearing loss. Cisplatin’s ototoxic effects are multifaceted, culminating in irreversible damage to the mechanosensory hair cells in the inner ear. Platinum drugs act on cancerous cells by forming nuclear DNA adducts, which may initiate signaling leading to cell cycle arrest or apoptosis. Moreover, it was reported that cisplatin may induce mitochondrial DNA damage in non-cancerous cells. Therefore, protecting mitochondria may alleviate cisplatin-induced insult to non-proliferating cells. Thus, it is important to identify agents that shield the mitochondria from cisplatin-induced insult without compromising the anti-tumor actions of the platinum-based drugs. In this study we tested the protective properties of mitochondrial division inhibitor, mdivi-1, a derivative of quinazolinone and a regulator of mitochondrial fission. Interestingly, it has been reported that mdivi-1 increases the apoptosis of cells that are resistant to cisplatin. The ability of mdivi-1 to protect hair cells against cisplatin-induced toxicity was evaluated in a fish model. Wild-type (Tübingen strain), cdh23 mutant, and transgenic pvalb3b::GFP zebrafish stably expressing GFP in the hair cells were used in this study. Larvae at 5–6 days post fertilization were placed in varying concentrations of cisplatin (50–200 μM) and/or mdivi-1 (1–10 μM) for 16 h. To evaluate hair cell’s viability the number of hair bundles per neuromast were counted. To assess hair cell function, we used the FM1-43 uptake assay and recordings of neuromast microphonic potentials. The results showed that mdivi-1 protected hair cells of lateral line neuromasts when they were challenged by 50 μM of cisplatin: viability of hair cells increased almost twice from 19% ± 1.8% to 36% ± 2.0% (p < 0.001). No protection was observed when higher concentrations of cisplatin were used. In addition, our data were in accord with previously reported results that functional mechanotransduction strongly potentiates cisplatin-induced hair cell toxicity. Together, our results suggest that mitochondrial protection may prevent cisplatin-induced damage to hair cells

Zebrafish models for the mechanosensory hair cell dysfunction in Usher syndrome 3 reveal that Clarin-1 is an essential hair bundle protein

UNLABELLED Usher syndrome type III (USH3) is characterized by progressive loss of hearing and vision, and varying degrees of vestibular dysfunction. It is caused by mutations that affect the human clarin-1 protein (hCLRN1), a member of the tetraspanin protein family. The missense mutation CLRN1(N48K), which affects a conserved N-glycosylation site in hCLRN1, is a common causative USH3 mutation among Ashkenazi Jews. The affected individuals hear at birth but lose that function over time. Here, we developed an animal model system using zebrafish transgenesis and gene targeting to provide an explanation for this phenotype. Immunolabeling demonstrated that Clrn1 localized to the hair cell bundles (hair bundles). The clrn1 mutants generated by zinc finger nucleases displayed aberrant hair bundle morphology with diminished function. Two transgenic zebrafish that express either hCLRN1 or hCLRN1(N48K) in hair cells were produced to examine the subcellular localization patterns of wild-type and mutant human proteins. hCLRN1 localized to the hair bundles similarly to zebrafish Clrn1; in contrast, hCLRN1(N48K) largely mislocalized to the cell body with a small amount reaching the hair bundle. We propose that this small amount of hCLRN1(N48K) in the hair bundle provides clarin-1-mediated function during the early stages of life; however, the presence of hCLRN1(N48K) in the hair bundle diminishes over time because of intracellular degradation of the mutant protein, leading to progressive loss of hair bundle integrity and hair cell function. These findings and genetic tools provide an understanding and path forward to identify therapies to mitigate hearing loss linked to the CLRN1 mutation. SIGNIFICANCE STATEMENT Mutations in the clarin-1 gene affect eye and ear function in humans. Individuals with the CLRN1(N48K) mutation are born able to hear but lose that function over time. Here, we develop an animal model system using zebrafish transgenesis and gene targeting to provide an explanation for this phenotype. This approach illuminates the role of clarin-1 and the molecular mechanism linked to the CLRN1(N48K) mutation in sensory hair cells of the inner ear. Additionally, the investigation provided an in vivo model to guide future drug discovery to rescue the hCLRN1(N48K) in hair cells