RESUMO
The endocochlear potential (EP) generated by the stria vascularis (SV) is necessary for hair cell mechanotransduction in the mammalian cochlea. We sought to create a model of EP dysfunction for the purposes of transcriptional analysis and treatment testing. By administering a single dose of cisplatin, a commonly prescribed cancer treatment drug with ototoxic side effects, to the adult mouse, we acutely disrupt EP generation. By combining these data with single cell RNA-sequencing findings, we identify transcriptional changes induced by cisplatin exposure, and by extension transcriptional changes accompanying EP reduction, in the major cell types of the SV. We use these data to identify gene regulatory networks unique to cisplatin treated SV, as well as the differentially expressed and druggable gene targets within those networks. Our results reconstruct transcriptional responses that occur in gene expression on the cellular level while identifying possible targets for interventions not only in cisplatin ototoxicity but also in EP dysfunction.
RESUMO
Dental epithelial stem cells give rise to four types of dental epithelial cells: inner enamel epithelium (IEE), outer enamel epithelium (OEE), stratum intermedium (SI), and stellate reticulum (SR). IEE cells further differentiate into enamel-forming ameloblasts, which play distinct roles, and are essential for enamel formation. These are conventionally classified by their shape, although their transcriptome and biological roles are yet to be fully understood. Here, we aimed to use single-cell RNA sequencing to clarify the heterogeneity of dental epithelial cell types. Unbiased clustering of 6,260 single cells from incisors of postnatal day 7 mice classified them into two clusters of ameloblast, IEE/OEE, SI/SR, and two mesenchymal populations. Secretory-stage ameloblasts expressed Amel and Enam were divided into Dspp + and Ambn + ameloblasts. Pseudo-time analysis indicated Dspp + ameloblasts differentiate into Ambn + ameloblasts. Further, Dspp and Ambn could be stage-specific markers of ameloblasts. Gene ontology analysis of each cluster indicated potent roles of cell types: OEE in the regulation of tooth size and SR in the transport of nutrients. Subsequently, we identified novel dental epithelial cell marker genes, namely Pttg1, Atf3, Cldn10, and Krt15. The results not only provided a resource of transcriptome data in dental cells but also contributed to the molecular analyses of enamel formation.
RESUMO
Dental enamel, the hardest tissue in the human body, is derived from dental epithelial cell ameloblast-secreted enamel matrices. Enamel mineralization occurs in a strictly synchronized manner along with ameloblast maturation in association with ion transport and pH balance, and any disruption of these processes results in enamel hypomineralization. G protein-coupled receptors (GPCRs) function as transducers of external signals by activating associated G proteins and regulate cellular physiology. Tissue-specific GPCRs play important roles in organ development, although their activities in tooth development remain poorly understood. The present results show that the adhesion GPCR Gpr115 (Adgrf4) is highly and preferentially expressed in mature ameloblasts and plays a crucial role during enamel mineralization. To investigate the in vivo function of Gpr115, knockout (Gpr115-KO) mice were created and found to develop hypomineralized enamel, with a larger acidic area because of the dysregulation of ion composition. Transcriptomic analysis also revealed that deletion of Gpr115 disrupted pH homeostasis and ion transport processes in enamel formation. In addition, in vitro analyses using the dental epithelial cell line cervical loop-derived dental epithelial (CLDE) cell demonstrated that Gpr115 is indispensable for the expression of carbonic anhydrase 6 (Car6), which has a critical role in enamel mineralization. Furthermore, an acidic condition induced Car6 expression under the regulation of Gpr115 in CLDE cells. Thus, we concluded that Gpr115 plays an important role in enamel mineralization via regulation of Car6 expression in ameloblasts. The present findings indicate a novel function of Gpr115 in ectodermal organ development and clarify the molecular mechanism of enamel formation.
Assuntos
Ameloblastos/metabolismo , Esmalte Dentário/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Células Cultivadas , Camundongos , Camundongos Knockout , Ratos , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/genéticaRESUMO
Sensory epithelia of the inner ear contain mechanosensory hair cells (HCs) and glia-like supporting cells (SCs), both of which are required for hearing and balance functions. Each of these cell types has unique responses to ototoxic and cytoprotective stimuli. Non-lethal heat stress in the mammalian utricle induces heat shock proteins (HSPs) and protects against ototoxic drug-induced hair cell death. Induction of HSPs in the utricle demonstrates cell-type specificity at the protein level, with HSP70 induction occurring primarily in SCs, while HSP32 (also known as heme oxygenase 1, HMOX1) is induced primarily in resident macrophages. Neither of these HSPs are robustly induced in HCs, suggesting that HCs may have little capacity for induction of stress-induced protective responses. To determine the transcriptional responses to heat shock of these different cell types, we performed cell-type-specific transcriptional profiling using the RiboTag method, which allows for immunoprecipitation (IP) of actively translating mRNAs from specific cell types. RNA-Seq differential gene expression analyses demonstrated that the RiboTag method identified known cell type-specific markers as well as new markers for HCs and SCs. Gene expression differences suggest that HCs and SCs exhibit differential transcriptional heat shock responses. The chaperonin family member Cct8 was significantly enriched only in heat-shocked HCs, while Hspa1l (HSP70 family), and Hspb1 and Cryab (HSP27 and HSP20 families, respectively) were enriched only in SCs. Together our data indicate that HCs exhibit a limited but unique heat shock response, and SCs exhibit a broader and more robust transcriptional response to protective heat stress.
RESUMO
Mechanosensory hair cells of the inner ear transduce auditory and vestibular sensory input. Hair cells are susceptible to death from a variety of stressors, including treatment with therapeutic drugs that have ototoxic side effects. There is a need for co-therapies to mitigate drug-induced ototoxicity, and we showed previously that induction of heat shock proteins (HSPs) protects against hair cell death and hearing loss caused by aminoglycoside antibiotics in mouse. Here, we utilized the library of integrated cellular signatures (LINCS) to identify perturbagens that induce transcriptional profiles similar to that of heat shock. Massively parallel sequencing of RNA (RNA-Seq) of heat shocked and control mouse utricles provided a heat shock gene expression signature that was used in conjunction with LINCS to identify candidate perturbagens, several of which were known to protect the inner ear. Our data indicate that LINCS is a useful tool to screen for compounds that generate specific gene expression signatures in the inner ear. Forty-two LINCS-identified perturbagens were tested for otoprotection in zebrafish, and three of these were protective. These compounds also induced the heat shock gene expression signature in mouse utricles, and one compound protected against aminoglycoside-induced hair cell death in whole organ cultures of utricles from adult mice.
RESUMO
BACKGROUND: Cell type-specific ribosome-pulldown has become an increasingly popular method for analysis of gene expression. It allows for expression analysis from intact tissues and monitoring of protein synthesis in vivo. However, while its utility has been assessed, technical aspects related to sequencing of these samples, often starting with a smaller amount of RNA, have not been reported. In this study, we evaluated the performance of five library prep protocols for ribosome-associated mRNAs when only 250 pg-4 ng of total RNA are used. RESULTS: We obtained total and RiboTag-IP RNA, in three biological replicates. We compared 5 methods of library preparation for Illumina Next Generation sequencing: NuGEN Ovation RNA-Seq system V2 Kit, TaKaRa SMARTer Stranded Total RNA-Seq Kit, TaKaRa SMART-Seq v4 Ultra Low Input RNA Kit, Illumina TruSeq RNA Library Prep Kit v2 and NEBNext® Ultra™ Directional RNA Library Prep Kit using slightly modified protocols each with 4 ng of total RNA. An additional set of samples was processed using the TruSeq kit with 70 ng, as a 'gold standard' control and the SMART-Seq v4 with 250 pg of total RNA. TruSeq-processed samples had the best metrics overall, with similar results for the 4 ng and 70 ng samples. The results of the SMART-Seq v4 processed samples were similar to TruSeq (Spearman correlation > 0.8) despite using lower amount of input RNA. All RiboTag-IP samples had an increase in the intronic reads compared with the corresponding whole tissue, suggesting that the IP captures some immature mRNAs. The SMARTer-processed samples had a higher representation of ribosomal and non-coding RNAs leading to lower representation of protein coding mRNA. The enrichment or depletion of IP samples compared to corresponding input RNA was similar across all kits except for SMARTer kit. CONCLUSION: RiboTag-seq can be performed successfully with as little as 250 pg of total RNA when using the SMART-Seq v4 kit and 4 ng when using the modified protocols of other library preparation kits. The SMART-Seq v4 and TruSeq kits resulted in the highest quality libraries. RiboTag IP RNA contains some immature transcripts.
Assuntos
Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Análise de Sequência de RNA/veterinária , Transcriptoma , Animais , Imunoprecipitação , Camundongos , Controle de Qualidade , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Ribossomos/genéticaRESUMO
The DNA-binding protein REST forms complexes with histone deacetylases (HDACs) to repress neuronal genes in non-neuronal cells. In differentiating neurons, REST is downregulated predominantly by transcriptional silencing. Here we report that post-transcriptional inactivation of REST by alternative splicing is required for hearing in humans and mice. We show that, in the mechanosensory hair cells of the mouse ear, regulated alternative splicing of a frameshift-causing exon into the Rest mRNA is essential for the derepression of many neuronal genes. Heterozygous deletion of this alternative exon of mouse Rest causes hair cell degeneration and deafness, and the HDAC inhibitor SAHA (Vorinostat) rescues the hearing of these mice. In humans, inhibition of the frameshifting splicing event by a novel REST variant is associated with dominantly inherited deafness. Our data reveal the necessity for alternative splicing-dependent regulation of REST in hair cells, and they identify a potential treatment for a group of hereditary deafness cases.
Assuntos
Surdez/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Processamento Alternativo/genética , Animais , Linhagem Celular , Éxons , Regulação da Expressão Gênica/genética , Células HEK293 , Células Ciliadas Auditivas/fisiologia , Audição/genética , Audição/fisiologia , Inibidores de Histona Desacetilases/metabolismo , Histona Desacetilases/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neurônios , Splicing de RNA/genética , Proteínas Repressoras/fisiologia , Fatores de Transcrição , Vorinostat/farmacologiaRESUMO
Protein-protein interactions (PPIs) regulate assembly of macromolecular complexes, yet remain challenging to study within the native cytoplasm where they normally exert their biological effect. Here we miniaturize the concept of affinity pulldown, a gold-standard in vitro PPI interrogation technique, to perform nanoscale pulldowns (NanoSPDs) within living cells. NanoSPD hijacks the normal process of intracellular trafficking by myosin motors to forcibly pull fluorescently tagged protein complexes along filopodial actin filaments. Using dual-color total internal reflection fluorescence microscopy, we demonstrate complex formation by showing that bait and prey molecules are simultaneously trafficked and actively concentrated into a nanoscopic volume at the tips of filopodia. The resulting molecular traffic jams at filopodial tips amplify fluorescence intensities and allow PPIs to be interrogated using standard epifluorescence microscopy. A rigorous quantification framework and software tool are provided to statistically evaluate NanoSPD data sets. We demonstrate the capabilities of NanoSPD for a range of nuclear and cytoplasmic PPIs implicated in human deafness, in addition to dissecting these interactions using domain mapping and mutagenesis experiments. The NanoSPD methodology is extensible for use with other fluorescent molecules, in addition to proteins, and the platform can be easily scaled for high-throughput applications.
Assuntos
Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Análise de Célula Única/métodos , Citoesqueleto de Actina/metabolismo , Movimento Celular , Proteínas de Fluorescência Verde/metabolismo , Proteínas Motores Moleculares , Miosinas/metabolismo , Domínios e Motivos de Interação entre Proteínas/fisiologia , Transporte Proteico , Pseudópodes/metabolismoRESUMO
Aminoglycoside ototoxicity involves the accumulation of antibiotic molecules in the inner ear hair cells and the subsequent degeneration of these cells. The exact route of entry of aminoglycosides into the hair cells in vivo is still unknown. Similar to other small organic cations, aminoglycosides could be brought into the cell by endocytosis or permeate through large non-selective cation channels, such as mechanotransduction channels or ATP-gated P2X channels. Here, we show that the aminoglycoside antibiotic gentamicin can enter mouse outer hair cells (OHCs) via TRPA1, non-selective cation channels activated by certain pungent compounds and by endogenous products of lipid peroxidation. Using conventional and perforated whole-cell patch clamp recordings, we found that application of TRPA1 agonists initiates inward current responses in wild-type OHCs, but not in OHCs of homozygous Trpa1 knockout mice. Similar responses consistent with the activation of non-selective cation channels were observed in heterologous cells transfected with mouse Trpa1. Upon brief activation with TRPA1 agonists, Trpa1-transfected cells become loaded with fluorescent gentamicin-Texas Red conjugate (GTTR). This uptake was not observed in mock-transfected or non-transfected cells. In mouse organ of Corti explants, TRPA1 activation resulted in the rapid entry of GTTR and another small cationic dye, FM1-43, in OHCs and some supporting cells, even when hair cell mechanotransduction was disrupted by pre-incubation in calcium-free solution. This TRPA1-mediated entry of GTTR and FM1-43 into OHCs was observed in wild-type but not in Trpa1 knockout mice and was not blocked by PPADS, a non-selective blocker of P2X channels. Notably, TRPA1 channels in mouse OHCs were activated by 4-hydroxynonenal, an endogenous molecule that is known to be generated during episodes of oxidative stress and accumulate in the cochlea after noise exposure. We concluded that TRPA1 channels may provide a novel pathway for the entry of aminoglycosides into OHCs.
Assuntos
Aminoglicosídeos/farmacocinética , Aminoglicosídeos/toxicidade , Gentamicinas/farmacocinética , Gentamicinas/toxicidade , Células Ciliadas Auditivas Externas , Canais de Potencial de Receptor Transitório/metabolismo , Aldeídos/farmacologia , Animais , Células COS , Cátions/farmacocinética , Chlorocebus aethiops , Inibidores de Cisteína Proteinase/farmacologia , Corantes Fluorescentes/farmacocinética , Genótipo , Células HEK293 , Células Ciliadas Auditivas Externas/efeitos dos fármacos , Células Ciliadas Auditivas Externas/metabolismo , Células Ciliadas Auditivas Externas/patologia , Humanos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Cultura de Órgãos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Técnicas de Patch-Clamp , Compostos de Piridínio/farmacocinética , Compostos de Amônio Quaternário/farmacocinética , Canal de Cátion TRPA1 , Canais de Potencial de Receptor Transitório/antagonistas & inibidores , Canais de Potencial de Receptor Transitório/genética , Xantenos/farmacocinéticaRESUMO
Targeted genome capture combined with next-generation sequencing was used to analyze 2.9 Mb of the DFNB79 interval on chromosome 9q34.3, which includes 108 candidate genes. Genomic DNA from an affected member of a consanguineous family segregating recessive, nonsyndromic hearing loss was used to make a library of fragments covering the DFNB79 linkage interval defined by genetic analyses of four pedigrees. Homozygosity for eight previously unreported variants in transcribed sequences was detected by evaluating a library of 402,554 sequencing reads and was later confirmed by Sanger sequencing. Of these variants, six were determined to be polymorphisms in the Pakistani population, and one was in a noncoding gene that was subsequently excluded genetically from the DFNB79 linkage interval. The remaining variant was a nonsense mutation in a predicted gene, C9orf75, renamed TPRN. Evaluation of the other three DFNB79-linked families identified three additional frameshift mutations, for a total of four truncating alleles of this gene. Although TPRN is expressed in many tissues, immunolocalization of the protein product in the mouse cochlea shows prominent expression in the taper region of hair cell stereocilia. Consequently, we named the protein taperin.
Assuntos
Cromossomos Humanos Par 9/genética , Surdez/genética , Mutação , Proteínas/genética , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Códon sem Sentido , Consanguinidade , Surdez/metabolismo , Feminino , Mutação da Fase de Leitura , Genes Recessivos , Células Ciliadas Auditivas/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Dados de Sequência Molecular , Fases de Leitura Aberta , Paquistão , Linhagem , Polimorfismo de Nucleotídeo Único , Proteínas/metabolismo , Distribuição TecidualRESUMO
Recessive mutations of MYO7A, encoding unconventional myosin VIIA, can cause either a deaf-blindness syndrome (type 1 Usher syndrome; USH1B) or nonsyndromic deafness (DFNB2). In our study, deafness segregating as a recessive trait in 24 consanguineous families showed linkage to markers for the DFNB2/USH1B locus on chromosome 11q13.5. A total of 23 of these families segregate USH1 due to 17 homozygous mutant MYO7A alleles, of which 14 are novel. One family segregated nonsyndromic hearing loss DFNB2 due to a novel three-nucleotide deletion in an exon of MYO7A (p.E1716del) encoding a region of the tail domain. We hypothesized that DFNB2 alleles of MYO7A have residual myosin VIIA. To address this question we investigated the effects of several mutant alleles by making green fluorescent protein (GFP) tagged cDNA expression constructs containing engineered mutations of mouse Myo7a at codons equivalent to pathogenic USH1B and DFNB2 alleles of human MYO7A. We show that in transfected mouse hair cells an USH1B mutant GFP-myosin VIIa does not localize properly to inner ear hair cell stereocilia. However, a GFP-myosin VIIa protein engineered to have an equivalent DFNB2 mutation to p.E1716del localizes correctly in transfected mouse hair cells. This finding is consistent with the hypothesis that p.E1716del causes a less severe phenotype (DFNB2) than the USH1B-associated alleles because the resulting protein retains some degree of normal function.
Assuntos
Surdez/genética , Dineínas/genética , Mutação , Miosinas/genética , Adulto , Alelos , Sequência de Aminoácidos , Animais , Composição de Bases , Cromossomos Humanos Par 11/genética , Consanguinidade , DNA Complementar/genética , Surdez/metabolismo , Surdez/fisiopatologia , Dineínas/química , Dineínas/metabolismo , Éxons , Feminino , Genes Recessivos , Ligação Genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células Ciliadas Auditivas Internas/metabolismo , Humanos , Cinética , Masculino , Camundongos , Pessoa de Meia-Idade , Modelos Moleculares , Dados de Sequência Molecular , Miosina VIIa , Miosinas/química , Miosinas/metabolismo , Linhagem , Fenótipo , Conformação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Transfecção , Síndromes de Usher/genética , Síndromes de Usher/metabolismo , Síndromes de Usher/fisiopatologiaRESUMO
Stereocilia are microvilli-derived mechanosensory organelles that are arranged in rows of graded heights on the apical surface of inner-ear hair cells. The 'staircase'-like architecture of stereocilia bundles is necessary to detect sound and head movement, and is achieved through differential elongation of the actin core of each stereocilium to a predetermined length. Abnormally short stereocilia bundles that have a diminished staircase are characteristic of the shaker 2 (Myo15a(sh2)) and whirler (Whrn(wi)) strains of deaf mice. We show that myosin-XVa is a motor protein that, in vivo, interacts with the third PDZ domain of whirlin through its carboxy-terminal PDZ-ligand. Myosin-XVa then delivers whirlin to the tips of stereocilia. Moreover, if green fluorescent protein (GFP)-Myo15a is transfected into hair cells of Myo15a(sh2) mice, the wild-type pattern of hair bundles is restored by recruitment of endogenous whirlin to the tips of stereocilia. The interaction of myosin-XVa and whirlin is therefore a key event in hair-bundle morphogenesis.
Assuntos
Cílios/ultraestrutura , Surdez/genética , Células Ciliadas Auditivas Internas/crescimento & desenvolvimento , Proteínas de Membrana/metabolismo , Miosinas/fisiologia , Actinas/metabolismo , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Proteínas de Fluorescência Verde , Células Ciliadas Auditivas Internas/ultraestrutura , Camundongos , Camundongos Mutantes , Modelos Biológicos , Proteínas Motores Moleculares/metabolismoRESUMO
Mutations in whirlin, a putative PDZ scaffold protein, have recently been shown to cause deafness and short cochlear hair cell stereocilia in whirler mice and recessive deafness (DFNB31) in humans. Through its PDZ domains, whirlin might organize a group of proteins into a functional complex required for stereocilia elongation. Identifying these protein partners will advance our understanding of the development of stereocilia and their function as mechanosensory organelles indispensable for normal hearing.
Assuntos
Surdez/genética , Células Ciliadas Auditivas/metabolismo , Proteínas de Membrana/genética , Animais , Surdez/metabolismo , Surdez/patologia , Células Ciliadas Auditivas/patologia , Humanos , Proteínas de Membrana/metabolismo , CamundongosRESUMO
Mutations of the gene encoding unconventional myosin XVa are associated with sensorineural deafness in humans (DFNB3) and shaker (Myo15sh2) mice. In deaf Myo15sh2/sh2 mice, stereocilia are short, nearly equal in length, and lack myosin XVa immunoreactivity. We previously reported that myosin XVa mRNA and protein are expressed in cochlear hair cells. We now show that in the mouse, rat, and guinea pig, endogenous myosin XVa localizes to the tips of the stereocilia of the cochlear and vestibular hair cells. Myosin XVa localization overlaps with the barbed ends of actin filaments and extends to the apical plasma membrane of the stereocilia. Gene gun-mediated transfection of mouse inner ear sensory epithelia explants shows selective accumulation of myosin XVa-GFP at the tips of stereocilia, confirming the localization of native myosin XVa. Expression in COS7 cells also reveals targeting of myosin XVa-GFP to the dynamic actin region at the tips of filopodia. In a wild-type mouse, during auditory and vestibular hair cell development, myosin XVa appears at the tips of stereocilia at the time when the hair bundle begins to develop its characteristic staircase pattern. We propose that myosin XVa is essential for the graded elongation of stereocilia during their functional maturation.
Assuntos
Células Ciliadas Auditivas Internas/metabolismo , Miosinas/metabolismo , Animais , Células COS , Cílios/metabolismo , Proteínas de Fluorescência Verde , Cobaias , Células Ciliadas Auditivas Internas/crescimento & desenvolvimento , Células Ciliadas Vestibulares/crescimento & desenvolvimento , Células Ciliadas Vestibulares/metabolismo , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Microscopia de Fluorescência , Mutação , Miosinas/genética , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
We have now identified seven MYO15A mutations that cause congenital profound neurosensory hearing loss and a possible hypomorphic allele of MYO15A associated with moderately-severe hearing loss in 1 of 8 SMS patients. Because myosin XVA is encoded by 66 exons, screening for mutations in hearing-impaired individuals is expensive and labor-intensive in comparison to a screen for mutations in GJB2 (Cx26), for example, which has only a single protein coding exon. Among consanguineous families segregating profound, congenital hearing loss from Pakistan, approximately 10% are consistent with linkage to DFNB3 (11 of 112 DFNB families). In one-half of these DFNB3 families, we found a homozygous mutation in 1 of the 66 exons of MYO15A [25]. This suggests that mutations of MYO15A are responsible for at least 5% of recessively inherited, profound hearing loss in Pakistan. However, without the benefit of a pre-screen for linkage to DFNB3, it will be a challenge to determine the extent to which mutations of MYO15A contribute to hereditary hearing loss among isolated cases and small families in other populations.