TMC1 and TMC2 Are Components of the Mechanotransduction Channel in Hair Cells of the Mammalian Inner Ear

SummarySensory transduction in auditory and vestibular hair cells requires expression of transmembrane channel-like (Tmc) 1 and 2 genes, but the function of these genes is unknown. To investigate the hypothesis that TMC1 and TMC2 proteins are components of the mechanosensitive ion channels that convert mechanical information into electrical signals, we recorded whole-cell and single-channel currents from mouse hair cells that expressed Tmc1, Tmc2, or mutant Tmc1. Cells that expressed Tmc2 had high calcium permeability and large single-channel currents, while cells with mutant Tmc1 had reduced calcium permeability and reduced single-channel currents. Cells that expressed Tmc1 and Tmc2 had a broad range of single-channel currents, suggesting multiple heteromeric assemblies of TMC subunits. The data demonstrate TMC1 and TMC2 are components of hair cell transduction channels and contribute to permeation properties. Gradients in TMC channel composition may also contribute to variation in sensory transduction along the tonotopic axis of the mammalian cochlea

Cell Type–Specific Transcriptome Analysis Reveals a Major Role for Zeb1 and miR-200b in Mouse Inner Ear Morphogenesis

Cellular heterogeneity hinders the extraction of functionally significant results and inference of regulatory networks from wide-scale expression profiles of complex mammalian organs. The mammalian inner ear consists of the auditory and vestibular systems that are each composed of hair cells, supporting cells, neurons, mesenchymal cells, other epithelial cells, and blood vessels. We developed a novel protocol to sort auditory and vestibular tissues of newborn mouse inner ears into their major cellular components. Transcriptome profiling of the sorted cells identified cell type–specific expression clusters. Computational analysis detected transcription factors and microRNAs that play key roles in determining cell identity in the inner ear. Specifically, our analysis revealed the role of the Zeb1/miR-200b pathway in establishing epithelial and mesenchymal identity in the inner ear. Furthermore, we detected a misregulation of the ZEB1 pathway in the inner ear of Twirler mice, which manifest, among other phenotypes, malformations of the auditory and vestibular labyrinth. The association of misregulation of the ZEB1/miR-200b pathway with auditory and vestibular defects in the Twirler mutant mice uncovers a novel mechanism underlying deafness and balance disorders. Our approach can be employed to decipher additional complex regulatory networks underlying other hearing and balance mouse mutants

A Noncoding Point Mutation of Zeb1 Causes Multiple Developmental Malformations and Obesity in Twirler Mice

Heterozygous Twirler (Tw) mice develop obesity and circling behavior associated with malformations of the inner ear, whereas homozygous Tw mice have cleft palate and die shortly after birth. Zeb1 is a zinc finger protein that contributes to mesenchymal cell fate by repression of genes whose expression defines epithelial cell identity. This developmental pathway is disrupted in inner ears of Tw/Tw mice. The purpose of our study was to comprehensively characterize the Twirler phenotype and to identify the causative mutation. The Tw/+ inner ear phenotype includes irregularities of the semicircular canals, abnormal utricular otoconia, a shortened cochlear duct, and hearing loss, whereas Tw/Tw ears are severely malformed with barely recognizable anatomy. Tw/+ mice have obesity associated with insulin-resistance and have lymphoid organ hypoplasia. We identified a noncoding nucleotide substitution, c.58+181G>A, in the first intron of the Tw allele of Zeb1 (Zeb1Tw). A knockin mouse model of c.58+181G>A recapitulated the Tw phenotype, whereas a wild-type knockin control did not, confirming the mutation as pathogenic. c.58+181G>A does not affect splicing but disrupts a predicted site for Myb protein binding, which we confirmed in vitro. In comparison, homozygosity for a targeted deletion of exon 1 of mouse Zeb1, Zeb1ΔEx1, is associated with a subtle abnormality of the lateral semicircular canal that is different than those in Tw mice. Expression analyses of E13.5 Twirler and Zeb1ΔEx1 ears confirm that Zeb1ΔEx1 is a null allele, whereas Zeb1Tw RNA is expressed at increased levels in comparison to wild-type Zeb1. We conclude that a noncoding point mutation of Zeb1 acts via a gain-of-function to disrupt regulation of Zeb1Tw expression, epithelial-mesenchymal cell fate or interactions, and structural development of the inner ear in Twirler mice. This is a novel mechanism underlying disorders of hearing or balance

Designing AAV Vectors for Monitoring the Subtle Calcium Fluctuations of Inferior Olive Network in vivo

Adeno-associated viral (AAV) vectors, used as vehicles for gene transfer into the brain, are a versatile and powerful tool of modern neuroscience that allow identifying specific neuronal populations, monitoring and modulating their activity. For consistent and reproducible results, the AAV vectors must be engineered so that they reliably and accurately target cell populations. Furthermore, transgene expression must be adjusted to sufficient and safe levels compatible with the physiology of studied cells. We undertook the effort to identify and validate an AAV vector that could be utilized for researching the inferior olivary (IO) nucleus, a structure gating critical timing-related signals to the cerebellum. By means of systematic construct generation and quantitative expression profiling, we succeeded in creating a viral tool for specific and strong transfection of the IO neurons without adverse effects on their physiology. The potential of these tools is demonstrated by expressing the calcium sensor GCaMP6s in adult mouse IO neurons. We could monitor subtle calcium fluctuations underlying two signatures of intrinsic IO activity: the subthreshold oscillations (STOs) and the variable-duration action potential waveforms both in-vitro and in-vivo. Further, we show that the expression levels of GCaMP6s allowing such recordings are compatible with the delicate calcium-based dynamics of IO neurons, inviting future work into the network dynamics of the olivo-cerebellar system in behaving animals

Amino acid 572 in TMC1: hot spot or critical functional residue for dominant mutations causing hearing impairment

Two different missense mutations, p.D572N and p.D572H, affecting the same nucleotide and codon of the TMC1 gene were earlier reported to cause autosomal dominant hearing impairment at locus DFNA36 in two North American families. No other dominant mutations of human TMC1 have been published. We ascertained a third North American family segregating autosomal dominant nonsyndromic hearing impairment at the DFNA36 locus. We identified the p.D572N mutation of TMC1 co-segregating with hearing loss in our study family. A comparative haplotype analysis of linked single nucleotide polymorphisms and short tandem repeats in the two families segregating p.D572N was not consistent with a founder effect. These findings can be explained in two ways. Either nucleotide 1714 is a hot spot for mutations or, alternatively, missense mutations at this site confer a specific pathogenic gain-of-function or dominant-negative effect

Esophageal xanthoma: presence of M2 macrophages suggests association with late inflammatory and reparative processes

Esophageal xanthoma is a rare lesion which is an asymptomatic small yellowish polyp, and most of the reported cases were solitary lesion. Histologically, aggregations of foam cells are found under the papillary hypertrophic squamous epithelium and the foam cells express CD68. The etiology of esophageal xanthoma is unknown. The focal irritation of the esophageal mucosa and infiltrated inflammatory cells are presumed to contribute to its pathogenesis. Although the pathogenesis may be associated with inflammation, the type and nature of the macrophages remain unclear. Here we report a 46-year-old male with esophageal xanthoma, which was incidentally found by endoscopy. Histologically, acute inflammation was not noted, and immunohistochemistry revealed that the foam cells seen in this case of esophageal xanthoma expressed increased levels of M2 macrophage markers. These findings suggest that esophageal xanthoma is associated with late inflammatory and reparative processes long after the initial inflammation of esophageal squamous epithelium

Co-expression of low-risk HPV E6/E7 and EBV LMP-1 leads to precancerous lesions by DNA damage

Abstract Background Low-risk human papillomavirus (HPV), such as types 6 and 11, is considered non-oncogenic, but these types have been detected in oral cancer tissue samples, suggesting their possible involvement in oral carcinogenesis. Because double infection of high-risk HPV and Epstein-Barr virus (EBV) is known to be involved in oral carcinogenesis, we hypothesized that low-risk HPV and EBV co-infection can transform the oral cells. To verify our hypothesis, we evaluated the transformation activity of cell lines expressing both low-risk HPV E6/E7 and EBV LMP-1. Methods We transduced HPV6, 11 and 16 E6/E7 genes and EBV LMP-1 gene into primary mouse embryonic fibroblasts. The cell lines were examined for indices of transformation activity such as proliferation, induction of DNA damage, resistance to apoptosis, anchorage-independent growth, and tumor formation in nude mice. To evaluate the signaling pathways involved in transformation, NF-κB and p53 activities were analyzed. We also assessed adhesion signaling molecules associated with anchorage-independent growth such as MMP-2, paxillin and Cat-1. Results Co-expression of low-risk HPV6 E6 and EBV LMP-1 showed increased cell proliferation, elevated NF-κB activity and reduced p53 induction. Moreover, co-expression of low-risk HPV6 E6 and EBV LMP-1 induced DNA damage, escaped from apoptosis under genotoxic condition and suppression of DNA damage response (DDR). Co-expression of low-risk HPV11 E6/E7 and EBV LMP-1 demonstrated similar results. However, it led to no malignant characteristics such as anchorage-independent growth, invasiveness and tumor formation in nude mice. Compared with the cells co-expressing high-risk HPV16 E6 and EBV LMP-1 that induce transformation, co-expression of low-risk HPV6 E6 and EBV LMP-1 was associated with low MMP-2, paxillin and Cat-1 expression. Conclusions The co-expression of low-risk HPV E6/E7 and EBV LMP-1 does not induce malignant transformation, but it allows accumulation of somatic mutations secondary to increased DNA damage and suppression of DDR. Thus, double infection of low-risk HPV and EBV could lead to precancerous lesions