Expression of a TMC6-TMC8-CIB1 heterotrimeric complex in lymphocytes is regulated by each of the components.

The TMC genes encode a set of homologous transmembrane proteins whose functions are not well understood. Biallelic mutations in either TMC6 or TMC8 are detected in more than half of cases of the pre-malignant skin disease epidermodysplasia verruciformis (EV). It is controversial whether EV induced by mutations in TMC6 or TMC8 originates from keratinocyte or lymphocyte defects. Quantification of TMC6 and TMC8 RNA levels in various organs revealed that lymphoid tissues have the highest levels of expression of both genes, and custom antibodies confirmed protein expression in mouse lymphocytes. To study the function of these proteins we generated mice with targeted deletion mutant alleles of Tmc6 or Tmc8 Either TMC6 or TMC8 deficiency induced a reduction in apparent molecular weight and/or amount of the other TMC molecule. Co-immunoprecipitation experiments indicated that TMC6 and TMC8 formed a protein complex in mouse and human T cells. MS and biochemical analysis demonstrated that TMC6 and TMC8 additionally interacted with the CIB1 protein to form TMC6-TMC8-CIB1 trimers. We demonstrated that TMC6 and TMC8 regulated CIB1 levels by protecting CIB1 from ubiquitination and proteasomal degradation. Reciprocally, CIB1 was needed for stabilizing TMC6 and TMC8 levels. These results suggest why inactivating mutations in any of the three human genes leads to similar clinical presentations. We also demonstrated that TMC6 and TMC8 levels are drastically lower and the proteins are less active in regulating CIB1 in keratinocytes than in T cells. Our study suggests that defects in lymphocytes may contribute to the etiology and pathogenesis of EV.

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

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.

NLRP3 mutation and cochlear autoinflammation cause syndromic and nonsyndromic hearing loss DFNA34 responsive to anakinra therapy.

The NLRP3 inflammasome is an intracellular innate immune sensor that is expressed in immune cells, including monocytes and macrophages. Activation of the NLRP3 inflammasome leads to IL-1ß secretion. Gain-of-function mutations of NLRP3 result in abnormal activation of the NLRP3 inflammasome, and cause the autosomal dominant systemic autoinflammatory disease spectrum, termed cryopyrin-associated periodic syndromes (CAPS). Here, we show that a missense mutation, p.Arg918Gln (c.2753G > A), of NLRP3 causes autosomal-dominant sensorineural hearing loss in two unrelated families. In family LMG446, hearing loss is accompanied by autoinflammatory signs and symptoms without serologic evidence of inflammation as part of an atypical CAPS phenotype and was reversed or improved by IL-1ß blockade therapy. In family LMG113, hearing loss segregates without any other target-organ manifestations of CAPS. This observation led us to explore the possibility that resident macrophage/monocyte-like cells in the cochlea can mediate local autoinflammation via activation of the NLRP3 inflammasome. The NLRP3 inflammasome can indeed be activated in resident macrophage/monocyte-like cells in the mouse cochlea, resulting in secretion of IL-1ß. This pathway could underlie treatable sensorineural hearing loss in DFNA34, CAPS, and possibly in a wide variety of hearing-loss disorders, such as sudden sensorineural hearing loss and Meniere's disease that are elicited by pathogens and processes that stimulate innate immune responses within the cochlea.

Transmembrane channel-like (TMC) genes are required for auditory and vestibular mechanosensation.

Mutations of the transmembrane channel-like 1 (TMC1) gene can cause dominant and recessive forms of deafness in humans and mice. TMC1 is one of eight mammalian TMC genes of unknown function. The multi-pass transmembrane topologic structure of the proteins they encode suggests roles as a receptor, transporter, channel, or pump. Tmc1 and the closely related Tmc2 gene are expressed in neurosensory hair cells of the auditory and vestibular end organs of the mouse inner ear. Recent studies have demonstrated that Tmc1 and Tmc2 are specifically required for mechanoelectrical transduction in hair cells. The exact role of these proteins in mechanoelectrical transduction is unknown. TMC1 and TMC2 are viable candidates for the mechanoelectrical transduction channel of hair cells, whose component molecules have eluded identification for over 30 years. We expect that studies of TMC proteins will yield insights into molecular components and mechanisms of mechanosensation in auditory and vestibular hair cells, as well as in other tissues and organs.

Slc26a4-insufficiency causes fluctuating hearing loss and stria vascularis dysfunction.

SLC26A4 mutations can cause a distinctive hearing loss phenotype with sudden drops and fluctuation in patients. Existing Slc26a4 mutant mouse lines have a profound loss of hearing and vestibular function, with severe inner ear malformations that do not model this human phenotype. In this study, we generated Slc26a4-insufficient mice by manipulation of doxycycline administration to a transgenic mouse line in which all Slc26a4 expression was under the control of doxycycline. Doxycycline was administered from conception to embryonic day 17.5, and then it was discontinued. Auditory brainstem response thresholds showed significant fluctuation of hearing loss from 1 through 3months of age. The endocochlear potential, which is required for inner ear sensory cell function, correlated with auditory brainstem response thresholds. We observed degeneration of stria vascularis intermediate cells, the cells that generate the endocochlear potential, but no other abnormalities within the cochlea. We conclude that fluctuations of hearing result from fluctuations of the endocochlear potential and stria vascularis dysfunction in Slc26a4-insufficient mouse ears. This model can now be used to test potential interventions to reduce or prevent sudden hearing loss or fluctuation in human patients. Our strategy to generate a hypomorphic mouse model utilizing the tet-on system will be applicable to other diseases in which a hypomorphic allele is needed to model the human phenotype.

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 (Zeb1(Tw)). 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, Zeb1(ΔEx1), 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 Zeb1(ΔEx1) ears confirm that Zeb1(ΔEx1) is a null allele, whereas Zeb1(Tw) 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 Zeb1(Tw) 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.

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.

Beethoven, a mouse model for dominant, progressive hearing loss DFNA36.

Despite recent progress in identifying genes underlying deafness, there are still relatively few mouse models of specific forms of human deafness. Here we describe the phenotype of the Beethoven (Bth) mouse mutant and a missense mutation in Tmc1 (transmembrane cochlear-expressed gene 1). Progressive hearing loss (DFNA36) and profound congenital deafness (DFNB7/B11) are caused by dominant and recessive mutations of the human ortholog, TMC1 (ref. 1), for which Bth and deafness (dn) are mouse models, respectively.

Dominant and recessive deafness caused by mutations of a novel gene, TMC1, required for cochlear hair-cell function.

Positional cloning of hereditary deafness genes is a direct approach to identify molecules and mechanisms underlying auditory function. Here we report a locus for dominant deafness, DFNA36, which maps to human chromosome 9q13-21 in a region overlapping the DFNB7/B11 locus for recessive deafness. We identified eight mutations in a new gene, transmembrane cochlear-expressed gene 1 (TMC1), in a DFNA36 family and eleven DFNB7/B11 families. We detected a 1.6-kb genomic deletion encompassing exon 14 of Tmc1 in the recessive deafness (dn) mouse mutant, which lacks auditory responses and has hair-cell degeneration. TMC1 and TMC2 on chromosome 20p13 are members of a gene family predicted to encode transmembrane proteins. Tmc1 mRNA is expressed in hair cells of the postnatal mouse cochlea and vestibular end organs and is required for normal function of cochlear hair cells.

Cytological characteristics of premalignant cervical epithelial lesions in postmenopausal women based on endocrine indices and parakeratosis.

OBJECTIVE: To identify useful cytological findings for detecting premalignant lesions in postmenopausal women, cervicovaginal smear samples were analyzed and compared between women with or without premalignant lesions based on endocrine indices and presence of parakeratosis (PK). METHODS: The cervicovaginal smear samples of postmenopausal women with premalignant lesions (n = 94) and those who were without (n = 344), who were diagnosed between 2012 and 2014 were retrieved and analyzed. Women cytologically diagnosed with malignancy or those with suspicion of malignancy were excluded from this study. Cytological endocrine indices, such as the maturation index (MI) and eosinophilic index (EI) and the prevalence of PK were compared between the groups and analyzed using the 2 × 2 χ2 test. The association of endocrine indices combined with the presence of PK and histological findings was also evaluated. RESULTS: Postmenopausal women with premalignant lesions had higher endocrine indices (EI of ≥11%; 65% vs. 43%, P < 0.01, f = 0.18) and a higher prevalence of PK positivity (PK ≥ 1; 46% vs. 7%, P < 0.01, f = 0.44) than those without lesions. Further analysis indicated that the combination of high EI and the presence of PK in postmenopausal women with cytological premalignant cases was highly associated with histological squamous intraepithelial lesions (SIL) (86% in women with premalignant lesions vs. 53% in those without; P = 0.01, f = 0.34). CONCLUSION: Our research demonstrated that high EI and PK positivity were correlated with SIL in postmenopausal women. These cytological findings could provide potential diagnostic clues for detecting dysplasia.

Selective binding and transport of protocadherin 15 isoforms by stereocilia unconventional myosins in a heterologous expression system.

During hair cell development, the mechanoelectrical transduction (MET) apparatus is assembled at the stereocilia tips, where it coexists with the stereocilia actin regulatory machinery. While the myosin-based tipward transport of actin regulatory proteins is well studied, isoform complexity and built-in redundancies in the MET apparatus have limited our understanding of how MET components are transported. We used a heterologous expression system to elucidate the myosin selective transport of isoforms of protocadherin 15 (PCDH15), the protein that mechanically gates the MET apparatus. We show that MYO7A selectively transports the CD3 isoform while MYO3A and MYO3B transports the CD2 isoform. Furthermore, MYO15A showed an insignificant role in the transport of PCDH15, and none of the myosins tested transport PCDH15-CD1. Our data suggest an important role for MYO3A, MYO3B, and MYO7A in the MET apparatus formation and highlight the intricate nature of MET and actin regulation during development and functional maturation of the stereocilia bundle.

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.

Systemic Fluorescent Gentamicin Enters Neonatal Mouse Hair Cells Predominantly Through Sensory Mechanoelectrical Transduction Channels.

Systemically administered aminoglycoside antibiotics can enter inner ear hair cells and trigger apoptosis. However, the in vivo route(s) by which aminoglycoside antibiotics enter hair cells remains controversial. Aminoglycosides can enter mouse hair cells by endocytosis or by permeation through transmembrane ion channels such as sensory mechanoelectrical transduction (MET) channels, transient receptor potential (TRP) channels, P2X channels, Piezo2-containing ion channels, or a combination of these routes. Transmembrane channel-like 1 (TMC1) and TMC2 are essential for sensory MET and appear to be the pore-forming components of sensory MET channels. The present study tested the hypothesis that systemic fluorescent gentamicin enters mouse hair cells predominantly through sensory MET channels. We employed Tmc1Δ, Tmc2Δ, and Tmc1::mCherry mice. In Tmc1::mCherry mice, the transgene was integrated on the X chromosome, resulting in mosaic expression of TMC1-mCherry in the hair cells of female heterozygous mice. After systemic administration of gentamicin-conjugated Texas Red (GTTR) into Tmc1Δ;Tmc2Δ mice and wild-type mice at postnatal day 4 (P4), robust GTTR fluorescence was detected in wild-type hair cells, whereas little or no GTTR fluorescence was detected in Tmc1Δ;Tmc2Δ hair cells. When GTTR was injected into developing mice at P0, P2, P4, or P6, the GTTR fluorescent intensity gradually increased from P0 to P4 in wild-type hair cells, whereas the intensity was stably low from P0 through P6 in Tmc1Δ;Tmc2Δ hair cells. The increase in the GTTR intensity coincided with the spatio-temporal onset of sensory MET in wild-type hair cells. In Tmc1::mCherry cochleae, only hair cells that showed a significant uptake of systemic GTTR took up FM1-43. Transmission electron microscopy could detect no disruption of normal endocytosis at the apical surface of Tmc1Δ;Tmc2Δ hair cells in vitro. These results provide substantial novel evidence that in vivo gentamicin enters neonatal mouse hair cells predominantly through sensory MET channels and not via endocytosis.

Multiparametric assessment of the impact of opsin expression and anesthesia on striatal cholinergic neurons and auditory brainstem activity.

Recent developments in genetic engineering have established murine models that permit the selective control of cholinergic neurons via optical stimulation. Despite copious benefits granted by these experimental advances, the sensory physiognomy of these organisms has remained poorly understood. Therefore, the present study evaluates sensory and neuronal response properties of animal models developed for the study of optically induced acetylcholine release regulation. Auditory brainstem responses, fluorescence imaging, and patch clamp recording techniques were used to assess the impact of viral infection, sex, age, and anesthetic agents across the ascending auditory pathway of ChAT-Cre and ChAT-ChR2(Ai32) mice. Data analyses revealed that neither genetic configuration nor adeno-associated viral infection alters the early stages of auditory processing or the cellular response properties of cholinergic neurons. However, anesthetic agent and dosage amount profoundly modulate the response properties of brainstem neurons. Last, analyses of age-related hearing loss in virally infected ChAT-Cre mice did not differ from those reported in wild type animals. This investigation demonstrates that ChAT-Cre and ChAT-ChR2(Ai32) mice are viable models for the study of cholinergic modulation in auditory processing, and it emphasizes the need for prudence in the selection of anesthetic procedures.

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.

K1 gene transformation activities in AIDS-related and classic type Kaposi's sarcoma: Correlation with clinical presentation.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes both AIDS-related Kaposi's sarcoma (KS) and classic KS, but their clinical presentations are different, and respective mechanisms remain to be elucidated. The KSHV K1 gene is reportedly involved in tumorigenesis through the immunoreceptor tyrosine-based activation motif (ITAM). Since we found the sequence variations in the K1 gene of KSHV isolated from AIDS-related KS and classic KS, we hypothesized that the transformation activity of the K1 gene contributes to the different clinical presentations. To evaluate our hypothesis, we compared the transformation activities of the K1 gene between AIDS-related KS and classic KS. We also analyzed ITAM activities and the downstream AKT and NF-κB. We found that the transformation activity of AIDS-related K1 was greater than that of classic K1, and that AIDS-related K1 induced higher ITAM activity than classic K1, causing more potent Akt and NF-κB activities. K1 downregulation by siRNA in AIDS-related K1 expressing cells induced a loss of transformation properties and decreased both Akt and NF-κB activities, suggesting a correlation between the transformation activity of K1 and ITAM signaling. Our study indicates that the increased transformation activity of AIDS-related K1 is associated with its clinical aggressiveness, whereas the weak transformation activity of classic type K1 is associated with a mild clinical presentation and spontaneous regression. The mechanism of spontaneous regression of classic KS may provide new therapeutic strategy to cancer.

Gradual Symmetric Progression of DFNA34 Hearing Loss Caused by an NLRP3 Mutation and Cochlear Autoinflammation.

OBJECTIVE: To characterize the audiometric phenotype of autosomal-dominant DFNA34 hearing loss (HL) caused by a missense substitution in the NLRP3 gene. NLRP3 encodes a critical component of the NLRP3 inflammasome that is activated in innate immune responses. STUDY DESIGN: This study was conducted under protocol 01-DC-0229 approved by the NIH Combined Neurosciences IRB. We performed medical and developmental history interviews and physical and audiological examinations of affected individuals with DFNA34 HL caused by the p.Arg918Gln mutation of NLRP3. We retrospectively reviewed audiological reports, when available, from other health care centers. SETTING: Federal biomedical research facility. SUBJECTS: Eleven members of a North American family segregating p.Arg918Gln. MAIN OUTCOME MEASURES: Pure-tone thresholds, rates of pure-tone threshold progression, and speech discrimination scores. RESULTS: Eight subjects had bilateral sensorineural HL with an onset in the late 2nd to 4th decade of life. Slowly progressive HL initially primarily affected high frequencies. Low and middle frequencies were affected with advancing age, resulting in moderate HL with a downsloping audiometric configuration. The average annual threshold deterioration was 0.9 to 1.5 dB/yr. Speech recognition scores ranging from 60 to 100% were consistent with cochlear, but not retrocochlear, etiology. Three subjects (16, 22, and 32 yr old) had normal hearing thresholds. CONCLUSION: DFNA34 HL has an onset during early adulthood and progresses approximately 1.2 dB/yr.

Molecular Signature of Tumors with Monoallelic 13q14 Deletion: a Case Series of Spindle Cell Lipoma and Genetically-Related Tumors Demonstrating a Link Between FOXO1 Status and p38 MAPK Pathway.

Spindle cell/pleomorphic lipomas (SCLs), cellular angiofibromas (CAFs) and mammary-type myofibroblastomas (MFBs) are rare benign mesenchymal tumors with monoallelic 13q14 deletion. They are predicted to have a common pathogenic mechanism due to shared similar histological and immunohistochemical features; however, pathological consequences of monoallelic 13q14 deletion remain unknown. We previously reported a CAF case with monoallelic 13q14 deletion in which the tumor expressed decreased levels of FOXO1 and RB1, both of which were encoded in 13q14, and increased reactive oxygen species (ROS) levels. We further demonstrated the activation of p38 mitogen-activated protein kinase (p38 MAPK) pathway induced by oxidative stress. We hypothesized that SCLs, CAFs and MFBs would share common molecular signatures involving FOXO1, ROS and p38 MAPK and that their expression patterns were different from those tumors without monoallelic 13q14 deletion such as solitary fibrous tumors (SFTs). We compared the expression levels of FOXO1, RB1, ROS markers and several signal transduction factors between SCLs and SFTs. SCLs expressed decreased levels of FOXO1 and RB1, whereas SFTs showed no change. Both tumor types exhibited increased markers of ROS; however, nuclear localization of phosphorylated p38 was significantly more frequent in SCLs than that in SFTs, suggesting p38 MAPK activation by oxidative stress. SFTs showed lower p38 MAPK activity and higher ß-catenin expression, implying that oxidative stress was caused by increased cellular proliferation stress. Finally, CAFs and MFBs showed changes similar to those observed in SCLs. Overall, tumors with monoallelic 13q14 deletion showed shared molecular signatures that might be associated with pathogenesis.

Tmc2 expression partially restores auditory function in a mouse model of DFNB7/B11 deafness caused by loss of Tmc1 function.

Mouse Tmc1 and Tmc2 are required for sensory transduction in cochlear and vestibular hair cells. Homozygous Tmc1∆/∆ mice are deaf, Tmc2∆/∆ mice have normal hearing, and double homozygous Tmc1∆/∆; Tmc2∆/∆ mice have deafness and profound vestibular dysfunction. These phenotypes are consistent with their different spatiotemporal expression patterns. Tmc1 expression is persistent in cochlear and vestibular hair cells, whereas Tmc2 expression is transient in cochlear hair cells but persistent in vestibular hair cells. On the basis of these findings, we hypothesized that persistent Tmc2 expression in mature cochlear hair cells could restore auditory function in Tmc1∆/∆ mice. To express Tmc2 in mature cochlear hair cells, we generated a transgenic mouse line, Tg[PTmc1::Tmc2], in which Tmc2 cDNA is expressed under the control of the Tmc1 promoter. The Tg[PTmc1::Tmc2] transgene slightly but significantly restored hearing in young Tmc1∆/∆ mice, though hearing thresholds were elevated with age. The elevation of hearing thresholds was associated with deterioration of sensory transduction in inner hair cells and loss of outer hair cell function. Although sensory transduction was retained in outer hair cells, their stereocilia eventually degenerated. These results indicate distinct roles and requirements for Tmc1 and Tmc2 in mature cochlear hair cells.

TMC1 Forms the Pore of Mechanosensory Transduction Channels in Vertebrate Inner Ear Hair Cells.

The proteins that form the permeation pathway of mechanosensory transduction channels in inner-ear hair cells have not been definitively identified. Genetic, anatomical, and physiological evidence support a role for transmembrane channel-like protein (TMC) 1 in hair cell sensory transduction, yet the molecular function of TMC proteins remains unclear. Here, we provide biochemical evidence suggesting TMC1 assembles as a dimer, along with structural and sequence analyses suggesting similarity to dimeric TMEM16 channels. To identify the pore region of TMC1, we used cysteine mutagenesis and expressed mutant TMC1 in hair cells of Tmc1/2-null mice. Cysteine-modification reagents rapidly and irreversibly altered permeation properties of mechanosensory transduction. We propose that TMC1 is structurally similar to TMEM16 channels and includes ten transmembrane domains with four domains, S4-S7, that line the channel pore. The data provide compelling evidence that TMC1 is a pore-forming component of sensory transduction channels in auditory and vestibular hair cells.