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1.
Development ; 148(14)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34228789

RESUMO

Sound transduction occurs in the hair bundle, the apical compartment of sensory hair cells in the inner ear. The hair bundle is formed of actin-based stereocilia aligned in rows of graded heights. It was previously shown that the GNAI-GPSM2 complex is part of a developmental blueprint that defines the polarized organization of the apical cytoskeleton in hair cells, including stereocilia distribution and elongation. Here, we report a role for multiple PDZ domain (MPDZ) protein during apical hair cell morphogenesis in mouse. We show that MPDZ is enriched at the hair cell apical membrane along with MAGUK p55 subfamily member 5 (MPP5/PALS1) and the Crumbs protein CRB3. MPDZ is required there to maintain the proper segregation of apical blueprint proteins, including GNAI-GPSM2. Loss of the blueprint coincides with misaligned stereocilia placement in Mpdz mutant hair cells, and results in permanently misshapen hair bundles. Graded molecular and structural defects along the cochlea can explain the profile of hearing loss in Mpdz mutants, where deficits are most severe at high frequencies.


Assuntos
Citoesqueleto/metabolismo , Células Ciliadas Auditivas/metabolismo , Domínios PDZ , Actinas/metabolismo , Animais , Cóclea/metabolismo , Orelha Interna/metabolismo , Perda Auditiva/metabolismo , Proteínas de Membrana , Camundongos , Estereocílios/metabolismo
2.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204426

RESUMO

The last decade has witnessed the identification of several families affected by hereditary non-syndromic hearing loss (NSHL) caused by mutations in the SMPX gene and the loss of function has been suggested as the underlying mechanism. In the attempt to confirm this hypothesis we generated an Smpx-deficient zebrafish model, pointing out its crucial role in proper inner ear development. Indeed, a marked decrease in the number of kinocilia together with structural alterations of the stereocilia and the kinocilium itself in the hair cells of the inner ear were observed. We also report the impairment of the mechanotransduction by the hair cells, making SMPX a potential key player in the construction of the machinery necessary for sound detection. This wealth of evidence provides the first possible explanation for hearing loss in SMPX-mutated patients. Additionally, we observed a clear muscular phenotype consisting of the defective organization and functioning of muscle fibers, strongly suggesting a potential role for the protein in the development of muscle fibers. This piece of evidence highlights the need for more in-depth analyses in search for possible correlations between SMPX mutations and muscular disorders in humans, thus potentially turning this non-syndromic hearing loss-associated gene into the genetic cause of dysfunctions characterized by more than one symptom, making SMPX a novel syndromic gene.


Assuntos
Orelha Interna/embriologia , Orelha Interna/metabolismo , Proteínas Musculares/deficiência , Músculos/embriologia , Músculos/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Desenvolvimento Embrionário , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Células Ciliadas Auditivas/metabolismo , Mecanotransdução Celular/genética , Desenvolvimento Muscular/genética , Organogênese/genética , Fenótipo , Transporte Proteico
3.
Cell Mol Life Sci ; 78(16): 6033-6049, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34274976

RESUMO

Melanocytes are pigmented cells residing mostly in the skin and hair follicles of vertebrates, where they contribute to colouration and protection against UV-B radiation. However, the spectrum of their functions reaches far beyond that. For instance, these pigment-producing cells are found inside the inner ear, where they contribute to the hearing function, and in the heart, where they are involved in the electrical conductivity and support the stiffness of cardiac valves. The embryonic origin of such extracutaneous melanocytes is not clear. We took advantage of lineage-tracing experiments combined with 3D visualizations and gene knockout strategies to address this long-standing question. We revealed that Schwann cell precursors are recruited from the local innervation during embryonic development and give rise to extracutaneous melanocytes in the heart, brain meninges, inner ear, and other locations. In embryos with a knockout of the EdnrB receptor, a condition imitating Waardenburg syndrome, we observed only nerve-associated melanoblasts, which failed to detach from the nerves and to enter the inner ear. Finally, we looked into the evolutionary aspects of extracutaneous melanocytes and found that pigment cells are associated mainly with nerves and blood vessels in amphibians and fish. This new knowledge of the nerve-dependent origin of extracutaneous pigment cells might be directly relevant to the formation of extracutaneous melanoma in humans.


Assuntos
Encéfalo/fisiologia , Orelha Interna/fisiologia , Coração/fisiologia , Meninges/fisiologia , Sistema Nervoso/fisiopatologia , Células de Schwann/fisiologia , Anfíbios/metabolismo , Anfíbios/fisiologia , Animais , Encéfalo/metabolismo , Linhagem da Célula/fisiologia , Orelha Interna/metabolismo , Desenvolvimento Embrionário/fisiologia , Feminino , Peixes/metabolismo , Peixes/fisiologia , Melanócitos/metabolismo , Melanócitos/fisiologia , Meninges/metabolismo , Camundongos , Sistema Nervoso/metabolismo , Gravidez , Receptor de Endotelina B/metabolismo , Células de Schwann/metabolismo
5.
Dev Biol ; 476: 200-208, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33864777

RESUMO

Nager syndrome is a rare human developmental disorder characterized by hypoplastic neural crest-derived craniofacial bones and limb defects. Mutations in SF3B4 gene, which encodes a component of the spliceosome, are a major cause for Nager. A review of the literature indicates that 45% of confirmed cases are also affected by conductive, sensorineural or mixed hearing loss. Conductive hearing loss is due to defective middle ear ossicles, which are neural crest derived, while sensorineural hearing loss typically results from defective inner ear or vestibulocochlear nerve, which are both derived from the otic placode. Animal model of Nager syndrome indicates that upon Sf3b4 knockdown cranial neural crest progenitors are depleted, which may account for the conductive hearing loss in these patients. To determine whether Sf3b4 plays a role in otic placode formation we analyzed the impact of Sf3b4 knockdown on otic development. Sf3b4-depleted Xenopus embryos exhibited reduced expression of several pan-placodal genes six1, dmrta1 and foxi4.1. We confirmed the dependence of placode genes expression on Sf3b4 function in animal cap explants expressing noggin, a BMP antagonist critical to induce placode fate in the ectoderm. Later in development, Sf3b4 morphant embryos had reduced expression of pax8, tbx2, otx2, bmp4 and wnt3a at the otic vesicle stage, and altered otic vesicle development. We propose that in addition to the neural crest, Sf3b4 is required for otic development, which may account for sensorineural hearing loss in Nager syndrome.


Assuntos
Perda Auditiva/genética , Disostose Mandibulofacial/genética , Fatores de Processamento de RNA/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Surdez/genética , Modelos Animais de Doenças , Orelha Interna/metabolismo , Ectoderma/metabolismo , Desenvolvimento Embrionário/genética , Gânglios Parassimpáticos/embriologia , Expressão Gênica/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Perda Auditiva/fisiopatologia , Disostose Mandibulofacial/metabolismo , Disostose Mandibulofacial/fisiopatologia , Crista Neural/embriologia , Fatores de Processamento de RNA/genética , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/metabolismo
6.
PLoS One ; 16(1): e0244661, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33513144

RESUMO

Amphimoschus is an extinct Eurasian ruminant genus, mostly recorded in Europe, without a close living relative and, hence, an unknown systematic position. This genus is known from around 50 localities from the late early to the middle Miocene. Two species were described during 180 years, but since their first description during the late 19th century and early 20th century, hardly any detailed taxonomic work has been done on the genus. Over the years, extensive collecting and excavating activities have enriched collections with more and more complete material of this still rare and enigmatic animal. Most interestingly, a number of skull remains have been unearthed and are promising in terms of providing phylogenetic information. In the present paper, we describe cranial material, the bony labyrinth, the dentition through 780 teeth and five skulls from different ontogenetic stages. We cannot find a clear morphometric distinction between the supposedly smaller and older species Amphimoschus artenensis and the supposedly younger and larger species A. ponteleviensis. Accordingly, we have no reason to retain the two species and propose, following the principle of priority (ICZN chapter 6 article 23), that only A. ponteleviensis Bourgeois, 1873 is valid. Our studies on the ontogenetic variation of Amphimoschus does reveal that the sagittal crest may increase in size and a supraorbital ridge may appear with age. Despite the abundant material, the family affiliation is still uncertain.


Assuntos
Artiodáctilos/anatomia & histologia , Fósseis/anatomia & histologia , Ruminantes/anatomia & histologia , Animais , Artiodáctilos/genética , Evolução Biológica , Dentição , Orelha Interna/anatomia & histologia , Orelha Interna/metabolismo , Europa (Continente) , Filogenia , Ruminantes/genética , Crânio/anatomia & histologia , Crânio/metabolismo
7.
Hum Mol Genet ; 29(22): 3691-3705, 2021 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-33326993

RESUMO

Hearing loss is a frequent sensory impairment in humans and genetic factors account for an elevated fraction of the cases. We have investigated a large family of five generations, with 15 reported individuals presenting non-syndromic, sensorineural, bilateral and progressive hearing loss, segregating as an autosomal dominant condition. Linkage analysis, using SNP-array and selected microsatellites, identified a region of near 13 cM in chromosome 20 as the best candidate to harbour the causative mutation. After exome sequencing and filtering of variants, only one predicted deleterious variant in the NCOA3 gene (NM_181659, c.2810C > G; p.Ser937Cys) fit in with our linkage data. RT-PCR, immunostaining and in situ hybridization showed expression of ncoa3 in the inner ear of mice and zebrafish. We generated a stable homozygous zebrafish mutant line using the CRISPR/Cas9 system. ncoa3-/- did not display any major morphological abnormalities in the ear, however, anterior macular hair cells showed altered orientation. Surprisingly, chondrocytes forming the ear cartilage showed abnormal behaviour in ncoa3-/-, detaching from their location, invading the ear canal and blocking the cristae. Adult mutants displayed accumulation of denser material wrapping the otoliths of ncoa3-/- and increased bone mineral density. Altered zebrafish swimming behaviour corroborates a potential role of ncoa3 in hearing loss. In conclusion, we identified a potential candidate gene to explain hereditary hearing loss, and our functional analyses suggest subtle and abnormal skeletal behaviour as mechanisms involved in the pathogenesis of progressive sensory function impairment.


Assuntos
Surdez/genética , Predisposição Genética para Doença , Perda Auditiva Neurossensorial/genética , Coativador 3 de Receptor Nuclear/genética , Adulto , Animais , Surdez/patologia , Modelos Animais de Doenças , Orelha Interna/metabolismo , Orelha Interna/patologia , Exoma/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patologia , Perda Auditiva Neurossensorial/patologia , Humanos , Masculino , Camundongos , Linhagem , Sequenciamento Completo do Exoma , Peixe-Zebra/genética
8.
Dev Biol ; 469: 160-171, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33131705

RESUMO

The inner ear comprises four epithelial domains: the cochlea, vestibule, semicircular canals, and endolymphatic duct/sac. These structures are segregated at embryonic day 13.5 (E13.5). However, these four anatomical structures remain undefined at E10.5. Here, we aimed to identify lineage-specific genes in the early developing inner ear using published data obtained from single-cell RNA-sequencing (scRNA-seq) of embryonic mice. We downloaded 5000 single-cell transcriptome data, named 'auditory epithelial trajectory', from the Mouse Organogenesis Cell Atlas. The dataset was supposed to include otic epithelial cells at E9.5-13.5. We projected the 5000 â€‹cells onto a two-dimensional space encoding the transcriptional state and visualised the pattern of otic epithelial cell differentiation. We identified 15 clusters, which were annotated as one of the four components of the inner ear epithelium using known genes that characterise the four different tissues. Additionally, we classified 15 clusters into sub-regions of the four inner ear components. By comparing transcriptomes between these 15 clusters, we identified several candidates of lineage-specific genes. Characterising these new candidate genes will help future studies about inner ear development.


Assuntos
Orelha Interna/embriologia , Orelha Interna/metabolismo , Animais , Diferenciação Celular/genética , Cóclea/metabolismo , Simulação por Computador , Orelha Interna/citologia , Embrião de Mamíferos/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Hibridização In Situ , Camundongos , Camundongos Endogâmicos ICR , RNA Mensageiro/metabolismo , RNA-Seq , Análise de Célula Única , Vestíbulo do Labirinto/metabolismo
9.
Int J Nanomedicine ; 15: 8921-8931, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33223827

RESUMO

Purpose: Drug delivery into the inner ear across the intact tympanic membrane (TM) has been a challenge in the treatment of inner ear disorders. In this study, nano-sized carriers were formulated for improving the non- invasive oto-topical delivery of caroverine for the treatment of tinnitus. Methods: Caroverine was loaded into two types of phospholipid-containing systems, namely, nano elastic vesicles (EVs) and phosphatidylcholine-based liquid crystalline nano-particles (PC-LCNPs). The prepared formulations were characterized for their drug loading, particle size, polydispersity index, zeta potential, morphological features by transmission electron microscopy (TEM), and physicochemical stability. In addition, comparative ex vivo transport study was carried out using rabbits' TM for both types of formulations. Results: The findings show a significant superiority of PC-LCNPs over the EVs formulations in the drug payload (1% and 0.25%, respectively), physical stability and the efficiency of permeation across rabbits' TM. The results showed a more than twofold increase in the cumulative drug flux values of PC-LCNPs (699.58 ± 100 µg/cm2) compared to the EVs (250 ± 45 µg/cm2) across the TM. Conclusion: The current study revealed the smart physicochemical properties of PC-LCNPs demonstrating the potential of this carrier as a new attractive candidate for improving the non-invasive oto-topical delivery of caroverine.


Assuntos
Portadores de Fármacos/química , Orelha Interna/metabolismo , Nanopartículas/química , Fosfatidilcolinas/química , Quinoxalinas/química , Quinoxalinas/metabolismo , Administração Cutânea , Animais , Transporte Biológico , Tamanho da Partícula , Quinoxalinas/administração & dosagem , Coelhos
10.
Sci Rep ; 10(1): 18225, 2020 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-33106554

RESUMO

Ancient DNA (aDNA) analyses necessitate the destructive sampling of archaeological material. Currently, the cochlea, part of the osseous inner ear located inside the petrous pyramid, is the most sought after skeletal element for molecular analyses of ancient humans as it has been shown to yield high amounts of endogenous DNA. However, destructive sampling of the petrous pyramid may not always be possible, particularly in cases where preservation of skeletal morphology is of top priority. To investigate alternatives, we present a survey of human aDNA preservation for each of ten skeletal elements in a skeletal collection from Medieval Germany. Through comparison of human DNA content and quality we confirm best performance of the petrous pyramid and identify seven additional sampling locations across four skeletal elements that yield adequate aDNA for most applications in human palaeogenetics. Our study provides a better perspective on DNA preservation across the human skeleton and takes a further step toward the more responsible use of ancient materials in human aDNA studies.


Assuntos
Osso e Ossos/metabolismo , DNA Antigo/química , DNA Antigo/isolamento & purificação , Orelha Interna/metabolismo , Osso Petroso/metabolismo , Preservação Biológica/métodos , Dente/metabolismo , Arqueologia , DNA Antigo/análise , Alemanha , História Medieval , Humanos
11.
Proc Natl Acad Sci U S A ; 117(40): 24837-24848, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32963095

RESUMO

The vertebrate inner ear, responsible for hearing and balance, is able to sense minute mechanical stimuli originating from an extraordinarily broad range of sound frequencies and intensities or from head movements. Integral to these processes is the tip-link protein complex, which conveys force to open the inner-ear transduction channels that mediate sensory perception. Protocadherin-15 and cadherin-23, two atypically large cadherins with 11 and 27 extracellular cadherin (EC) repeats, are involved in deafness and balance disorders and assemble as parallel homodimers that interact to form the tip link. Here we report the X-ray crystal structure of a protocadherin-15 + cadherin-23 heterotetrameric complex at 2.9-Å resolution, depicting a parallel homodimer of protocadherin-15 EC1-3 molecules forming an antiparallel complex with two cadherin-23 EC1-2 molecules. In addition, we report structures for 10 protocadherin-15 fragments used to build complete high-resolution models of the monomeric protocadherin-15 ectodomain. Molecular dynamics simulations and validated crystal contacts are used to propose models for the complete extracellular protocadherin-15 parallel homodimer and the tip-link bond. Steered molecular dynamics simulations of these models suggest conditions in which a structurally diverse and multimodal protocadherin-15 ectodomain can act as a stiff or soft gating spring. These results reveal the structural determinants of tip-link-mediated inner-ear sensory perception and elucidate protocadherin-15's structural and adhesive properties relevant in disease.


Assuntos
Percepção Auditiva , Caderinas/química , Caderinas/metabolismo , Caderinas/genética , Dimerização , Orelha Interna/metabolismo , Audição , Humanos , Simulação de Dinâmica Molecular , Equilíbrio Postural , Ligação Proteica , Conformação Proteica , Domínios Proteicos
12.
Neural Plast ; 2020: 8898811, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32774357

RESUMO

Alternative splicing plays a pivotal role in modulating the function of eukaryotic proteins. In the inner ear, many genes undergo alternative splicing, and errors in this process lead to hearing loss. Cadherin 23 (CDH23) forms part of the so-called tip links, which are indispensable for mechanoelectrical transduction (MET) in the hair cells. Cdh23 gene contains 69 exons, and exon 68 is subjected to alternative splicing. Exon 68 of the Cdh23 gene is spliced into its mRNA only in a few cell types including hair cells. The mechanism responsible for the alternative splicing of Cdh23 exon 68 remains elusive. In the present work, we performed a cell-based screening to look for splicing factors that regulate the splicing of Cdh23 exon 68. RBM24 and RBM38 were identified to enhance the inclusion of Cdh23 exon 68. The splicing of Cdh23 exon 68 is affected in Rbm24 knockdown or knockout cells. Moreover, we also found that PTBP1 inhibits the inclusion of Cdh23 exon 68. Taken together, we show here that alternative splicing of Cdh23 exon 68 is regulated by RBM24, RBM38, and PTBP1.


Assuntos
Processamento Alternativo , Caderinas/genética , Orelha Interna/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteínas de Ligação a RNA/genética , Animais , Linhagem Celular , Chlorocebus aethiops , Éxons , Camundongos
13.
Int J Mol Sci ; 21(16)2020 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-32806650

RESUMO

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, is involved in multiple and fundamental functions of the central and peripheral nervous systems including sensory organs. Despite recent advances in knowledge on the functional significance of BDNF and TrkB in the regulation of the acoustic system of mammals, the localization of BDNF/TrkB system in the inner ear of zebrafish during development, is not well known. Therefore, the goal of the present study is to analyze the age-dependent changes using RT-PCR, Western Blot and single and double immunofluorescence of the BDNF and its specific receptor in the zebrafish inner ear. The results showed the mRNA expression and the cell localization of BDNF and TrkB in the hair cells of the crista ampullaris and in the neuroepithelium of the utricle, saccule and macula lagena, analyzed at different ages. Our results demonstrate that the BDNF/TrkB system is present in the sensory cells of the inner ear, during whole life. Therefore, this system might play a key role in the development and maintenance of the hair cells in adults, suggesting that the zebrafish inner ear represents an interesting model to study the involvement of the neurotrophins in the biology of sensory cells.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Orelha Interna/metabolismo , Receptor trkB/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Orelha Interna/anatomia & histologia , Orelha Interna/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Larva/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor trkB/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
14.
Elife ; 92020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32352377

RESUMO

The sox2 expressing (sox2+) progenitors in adult mammalian inner ear lose the capacity to regenerate while progenitors in the zebrafish lateral line are able to proliferate and regenerate damaged HCs throughout lifetime. To mimic the HC damage in mammals, we have established a zebrafish severe injury model to eliminate both progenitors and HCs. The atoh1a expressing (atoh1a+) HC precursors were the main population that survived post severe injury, and gained sox2 expression to initiate progenitor regeneration. In response to severe injury, yap was activated to upregulate lin28a transcription. Severe-injury-induced progenitor regeneration was disabled in lin28a or yap mutants. In contrary, overexpression of lin28a initiated the recovery of sox2+ progenitors. Mechanistically, microRNA let7 acted downstream of lin28a to activate Wnt pathway for promoting regeneration. Our findings that lin28a is necessary and sufficient to regenerate the exhausted sox2+ progenitors shed light on restoration of progenitors to initiate HC regeneration in mammals.


Assuntos
Sistema da Linha Lateral/metabolismo , Regeneração Nervosa/fisiologia , Receptores Notch/metabolismo , Regeneração/fisiologia , Animais , Proliferação de Células/fisiologia , Orelha Interna/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células Ciliadas Auditivas/fisiologia , Fatores de Transcrição/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
15.
Cells ; 9(5)2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32455934

RESUMO

Nonsyndromic hearing loss (NSHL) is of great clinical importance, and mutations in the GJB2 gene and the encoded human CONNEXIN 26 (CX26) protein play important roles in the genetic pathogenesis. The CX26 p.R184Q mutation was shown to be a dominant-negative effect in our previous study. Previously, we also demonstrated that zebrafish Cx30.3 is orthologous to human CX26. In the present study, we established transgenic zebrafish models with mutated Cx30.3 specifically expressed in the supporting cells of zebrafish inner ears driven by the agr2 promoter, to demonstrate and understand the mechanism by which the human CX26 R.184 mutation causes NSHL. Our results indicated that significant structural changes in the inner ears of transgenic lines with mutations were measured and compared to wild-type zebrafish. Simultaneously, significant alterations of transgenic lines with mutations in swimming behavior were analyzed with the zebrafish behavioral assay. This is the first study to investigate the functional results of the CX26 p.R184Q mutation with in vivo disease models. Our work supports and confirms the pathogenic role of the CX26 p.R184Q mutation in NSHL, with a hypothesized mechanism of altered interaction among amino acids in the connexins.


Assuntos
Conexina 26/genética , Conexinas/genética , Surdez/genética , Mutação/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Comportamento Animal , Bioensaio , Conexinas/química , Modelos Animais de Doenças , Orelha Interna/metabolismo , Orelha Interna/patologia , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Proteínas Mutantes/química
16.
Nucleic Acids Res ; 48(9): 5065-5080, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32249312

RESUMO

Disabling hearing loss impacts ∼466 million individuals worldwide with 34 million children affected. Gene and pharmacotherapeutic strategies to rescue auditory function in mouse models of human deafness are most effective when administered before hearing onset, after which therapeutic efficacy is significantly diminished or lost. We hypothesize that preemptive correction of a mutation in the fetal inner ear prior to maturation of the sensory epithelium will optimally restore sensory function. We previously demonstrated that transuterine microinjection of a splice-switching antisense oligonucleotide (ASO) into the amniotic cavity immediately surrounding the embryo on embryonic day 13-13.5 (E13-13.5) corrected pre-mRNA splicing in the juvenile Usher syndrome type 1c (Ush1c) mouse mutant. Here, we show that this strategy only marginally rescues hearing and partially rescues vestibular function. To improve therapeutic outcomes, we microinjected ASO directly into the E12.5 inner ear. A single intra-otic dose of ASO corrects harmonin RNA splicing, restores harmonin protein expression in sensory hair cell bundles, prevents hair cell loss, improves hearing sensitivity, and ameliorates vestibular dysfunction. Improvements in auditory and vestibular function were sustained well into adulthood. Our results demonstrate that an ASO pharmacotherapeutic administered to a developing organ system in utero preemptively corrects pre-mRNA splicing to abrogate the disease phenotype.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas do Citoesqueleto/genética , Surdez/congênito , Surdez/tratamento farmacológico , Oligonucleotídeos Antissenso/uso terapêutico , Vestíbulo do Labirinto/fisiopatologia , Âmnio , Animais , Limiar Auditivo/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Surdez/genética , Surdez/fisiopatologia , Orelha Interna/efeitos dos fármacos , Orelha Interna/metabolismo , Feto , Células Ciliadas Auditivas/efeitos dos fármacos , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/ultraestrutura , Camundongos , Microinjeções , Mutação , Oligonucleotídeos Antissenso/administração & dosagem , Splicing de RNA/efeitos dos fármacos , Vestíbulo do Labirinto/efeitos dos fármacos
17.
Mol Brain ; 13(1): 29, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-32127020

RESUMO

Tsukushi (TSK)-a small, secreted, leucine-rich-repeat proteoglycan-interacts with and regulates essential cellular signaling cascades. However, its functions in the mouse inner ear are unknown. In this study, measurement of auditory brainstem responses, fluorescence microscopy, and scanning electron microscopy revealed that TSK deficiency in mice resulted in the formation of abnormal stereocilia in the inner hair cells and hearing loss but not in the loss of these cells. TSK accumulated in nonprosensory regions during early embryonic stages and in both nonprosensory and prosensory regions in late embryonic stages. In adult mice, TSK was localized in the organ of Corti, spiral ganglion cells, and the stria vascularis. Moreover, loss of TSK caused dynamic changes in the expression of key genes that drive the differentiation of the inner hair cells in prosensory regions. Finally, our results revealed that TSK interacted with Sox2 and BMP4 to control stereocilia formation in the inner hair cells. Hence, TSK appears to be an essential component of the molecular pathways that regulate inner ear development.


Assuntos
Orelha Interna/embriologia , Orelha Interna/metabolismo , Proteoglicanas/metabolismo , Animais , Proteína Morfogenética Óssea 4/metabolismo , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas Internas/metabolismo , Audição , Ligamentos/metabolismo , Camundongos Knockout , Proteoglicanas/deficiência , Proteoglicanas/genética , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Gânglio Espiral da Cóclea/metabolismo , Estereocílios/metabolismo
18.
Dev Biol ; 462(1): 74-84, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32147304

RESUMO

The five vestibular organs of the inner ear derive from patches of prosensory cells that express the transcription factor SOX2 and the Notch ligand JAG1. Previous work suggests that JAG1-mediated Notch signaling is both necessary and sufficient for prosensory formation and that the separation of developing prosensory patches is regulated by LMX1a, which antagonizes Notch signaling. We used an inner ear-specific deletion of the Rbpjκ gene in which Notch signaling is progressively lost from the inner ear to show that Notch signaling, is continuously required for the maintenance of prosensory fate. Loss of Notch signaling in prosensory patches causes them to shrink and ultimately disappear. We show this loss of prosensory fate is not due to cell death, but rather to the conversion of prosensory tissue into non-sensory tissue that expresses LMX1a. Notch signaling is therefore likely to stabilize, rather than induce prosensory fate.


Assuntos
Orelha Interna/embriologia , Proteína Jagged-1/metabolismo , Receptores Notch/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular , Orelha Interna/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Células Ciliadas Auditivas Internas/citologia , Proteína Jagged-1/genética , Proteínas com Homeodomínio LIM/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Organogênese/fisiologia , Receptores Notch/fisiologia , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo
19.
J Nanobiotechnology ; 18(1): 53, 2020 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-32192504

RESUMO

BACKGROUND: Ototoxicity is one of the major side effects of platinum-based chemotherapy, especially cisplatin therapy. To date, no FDA approved agents to alleviate or prevent this ototoxicity are available. However, ototoxicity is generally believed to be produced by excessive generation of reactive oxygen species (ROS) in the inner ear, thus leading to the development of various antioxidants, which act as otoprotective agents. Astaxanthin (ATX) is an interesting candidate in the development of new therapies for preventing and treating oxidative stress-related pathologies, owing to its unique antioxidant capacity. METHODS AND RESULTS: In this study, we aimed to evaluate the potential antioxidant properties of ATX in the inner ear by using the HEI-OC1 cell line, zebrafish, and guinea pigs. Because ATX has poor solubility and cannot pass through round window membranes (RWM), we established lipid-polymer hybrid nanoparticles (LPN) for loading ATX. The LPN enabled ATX to penetrate RWM and maintain concentrations in the perilymph in the inner ear for 24 h after a single injection. ATX-LPN were found to have favorable biocompatibility and to strongly affect cisplatin-induced generation of ROS, on the basis of DCFHDA staining in HEI-OC1 cells. JC-1 and MitoTracker Green staining suggested that ATX-LPN successfully reversed the decrease in mitochondrial membrane potential induced by cisplatin in vitro and rescued cells from early stages of apoptosis, as demonstrated by FACS stained with Annexin V-FITC/PI. Moreover, ATX-LPN successfully attenuated OHC losses in cultured organ of Corti and animal models (zebrafish and guinea pigs) in vivo. In investigating the protective mechanism of ATX-LPN, we found that ATX-LPN decreased the expression of pro-apoptotic proteins (caspase 3/9 and cytochrome-c) and increased expression of the anti-apoptotic protein Bcl-2. In addition, the activation of JNK induced by CDDP was up-regulated and then decreased after the administration of ATX-LPN, while P38 stayed unchanged. CONCLUSIONS: To best of our knowledge, this is first study concluded that ATX-LPN as a new therapeutic agent for the prevention of cisplatin-induced ototoxicity.


Assuntos
Nanopartículas/química , Polímeros/farmacologia , Xantofilas/farmacologia , Animais , Antioxidantes , Apoptose/efeitos dos fármacos , Caspase 3 , Linhagem Celular , Cisplatino/farmacologia , Orelha Interna/metabolismo , Cobaias , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Modelos Animais , Polímeros/química , Proteínas Proto-Oncogênicas c-bcl-2 , Espécies Reativas de Oxigênio/metabolismo , Xantofilas/química , Peixe-Zebra
20.
Adv Exp Med Biol ; 1218: 129-157, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32060875

RESUMO

Notch signalling is a major regulator of cell fate decisions and tissue patterning in metazoans. It is best known for its role in lateral inhibition, whereby Notch mediates competitive interactions between cells to limit adoption of a given developmental fate. However, it can also function by lateral induction, a cooperative mode of action that was originally described during the patterning of the Drosophila wing disc and creates boundaries or domains of cells of the same character. In this chapter, we introduce these two signalling modes and explain how they contribute to distinct aspects of the development and regeneration of the vertebrate inner ear, the organ responsible for the perception of sound and head movements. We discuss some of the factors that could influence the context-specific outcomes of Notch signalling in the inner ear and the ongoing efforts to target this pathway for the treatment of hearing loss and vestibular dysfunction.


Assuntos
Diferenciação Celular , Orelha Interna/embriologia , Orelha Interna/fisiologia , Receptores Notch/metabolismo , Regeneração , Transdução de Sinais , Animais , Orelha Interna/citologia , Orelha Interna/metabolismo , Perda Auditiva/metabolismo , Perda Auditiva/fisiopatologia , Humanos
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