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We developed a rat dorsal root ganglion (DRG)-derived sensory nerve organotypic model by culturing DRG explants on an organoid culture device. With this method, a large number of organotypic cultures can be produced simultaneously with high reproducibility simply by seeding DRG explants derived from rat embryos. Unlike previous DRG explant models, this organotypic model consists of a ganglion and an axon bundle with myelinated A fibers, unmyelinated C fibers, and stereo-myelin-forming nodes of Ranvier. The model also exhibits Ca2+ signaling in cell bodies in response to application of chemical stimuli to nerve terminals. Further, axonal transection increases the activating transcription factor 3 mRNA level in ganglia. Axons and myelin are shown to regenerate 14 days following transection. Our sensory organotypic model enables analysis of neuronal excitability in response to pain stimuli and tracking of morphological changes in the axon bundle over weeks.
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Axones , Ganglios Espinales , Sistemas Microfisiológicos , Animales , Ratas , Factor de Transcripción Activador 3 , Axones/fisiología , Axones/metabolismo , Señalización del Calcio , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Vaina de Mielina/fisiología , Vaina de Mielina/metabolismo , Organoides/metabolismo , Nervios Periféricos/metabolismo , Ratas Sprague-Dawley , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/fisiologíaRESUMEN
The pathophysiology of laryngopharyngeal reflux (LPR) and its impact on the vocal fold is not well understood, but may involve acid damage to vocal fold barrier functions. Two different components encompass vocal fold barrier function: the mucus barrier and tight junctions. Mucus retained on epithelial microprojections protects the inside of the vocal fold by neutralizing acidic damage. Tight junctions control permeability between cells. Here we developed an in vitro experimental system to evaluate acidic injury and repair of vocal fold barrier functions. We first established an in vitro model of rat vocal fold epithelium that could survive at least one week after barrier function maturation. The model enabled repeated evaluation of the course of vocal fold repair processes. Then, an injury experiment was conducted in which vocal fold cells were exposed to a 5-min treatment with acidic pepsin that injured tight junctions and cell surface microprojections. Both of them healed within one day of injury. Comparing vocal fold cells treated with acid alone with cells treated with acidic pepsin showed that acidic pepsin had a stronger effect on intercellular permeability than acid alone, whereas pepsin had little effect on microprojections. This result suggests that the proteolytic action of pepsin has a larger effect on protein-based tight junctions than on phospholipids in microprojections. This experimental system could contribute to a better understanding of vocal fold repair processes after chemical or physical injuries, as well as voice problems due to LPR pathogenesis.
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Cassidine leaf beetles are associated with genome-reduced symbiotic bacteria Stammera involved in pectin digestion. Stammera cells appear to be harbored in paired symbiotic organs located at the foregut-midgut junction either intracellularly or extracellularly, whereas the symbiont is extracellular in the ovary-accessory glands of adult females and during caplet transmission in eggs. However, using fluorescence and electron microscopy, an intracellular symbiotic configuration of Stammera was observed in Notosacantha species. Detailed inspection of other cassidine species revealed fragmented cell membrane and cytoplasm of the symbiotic organs, wherein Stammera cells are in an intermediate status between intracellularity and extracellularity. We also identified a mitochondria-rich region adjacent to the symbiont-filled region and well-developed muscle fibers surrounding the whole symbiotic organ. Based on these observations, we discuss why the Stammera genome has been reduced so drastically and how symbiont-derived pectinases are produced and supplied to the host's alimentary tract for plant cell wall digestion.
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For mucociliary clearance of pathogens, tracheal multiciliated epithelial cells (MCCs) organize coordinated beating of cilia, which originate from basal bodies (BBs) with basal feet (BFs) on one side. To clarify the self-organizing mechanism of coordinated intracellular BB-arrays composed of a well-ordered BB-alignment and unidirectional BB-orientation, determined by the direction of BB to BF, we generated double transgenic mice with GFP-centrin2-labeled BBs and mRuby3-Cep128-labeled BFs for long-term, high-resolution, dual-color live-cell imaging in primary-cultured tracheal MCCs. At early timepoints of MCC differentiation, BB-orientation and BB-local alignment antecedently coordinated in an apical microtubule-dependent manner. Later during MCC differentiation, fluctuations in BB-orientation were restricted, and locally aligned BB-arrays were further coordinated to align across the entire cell (BB-global alignment), mainly in an apical intermediate-sized filament-lattice-dependent manner. Thus, the high coordination of the BB-array was established for efficient mucociliary clearance as the primary defense against pathogen infection, identifying apical cytoskeletons as potential therapeutic targets.
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Cuerpos Basales , Citoesqueleto , Ratones , Animales , Microtúbulos , Cilios , Células EpitelialesRESUMEN
The maintenance of planar polarity in airway multiciliated cells (MCCs) has been poorly characterized. We recently reported that the direction of ciliary beating in a surgically inverted tracheal segment remained inverted beyond the time required for the turnover of cells, without adjustment to global distal-to-proximal polarity. We hypothesized that the local maintenance of tissue-level polarity occurs via locally reproduced cells. To provide further insight regarding this hypothetical property, we performed allotransplantation of an inverted tracheal segment between wild-type (donor) and tdTomato-expressing (host) rats, with and without scratching the mucosa of the transplants. The origin of cells in the transplants was assessed using tdTomato-specific immunostaining. Ciliary movement and structures were observed by high-speed video and electron microscopy to analyze MCC orientations. Variabilities in the orientations of closely and distantly located MCCs were analyzed to evaluate the local- and broad-scale coordination of polarity, respectively. The epithelium was maintained by donor-derived cells in the non-scratched inverted transplant over 6 months, beyond one cycle of turnover. The inverted orientation of MCCs was also maintained throughout the non-scratched transplant. MCCs regenerated in the scratched transplant were derived from the host and exhibited diverse orientations across the transplant. However, the orientations of adjacent regenerated MCCs were often coordinated, indicating that airway MCCs can locally coordinate their orientations. A steady-state airway may maintain MCC orientation by locally reproducing MCCs via the local coordination of polarity. This local coordination enables the formation and maintenance of tissue-level polarity in small regions after mucosal injury.
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Cancer cells generally exhibit increased iron uptake, which contributes to their abnormal growth and metastatic ability. Iron chelators have thus recently attracted attention as potential anticancer agents. Here, we show that deferriferrichrysin (Dfcy), a natural product from Aspergillus oryzae acts as an iron chelator to induce paraptosis (a programmed cell death pathway characterized by ER dilation) in MCF-7 human breast cancer cells and H1299 human lung cancer cells. We first examined the anticancer efficacy of Dfcy in cancer cells and found that Dfcy induced ER dilation and reduced the number of viable cells. Extracellular signal-related kinase (ERK) was activated by Dfcy treatment, and the MEK inhibitor U0126, a small molecule commonly used to inhibit ERK activity, prevented the increase in ER dilation in Dfcy-treated cells. Concomitantly, the decrease in the number of viable cells upon treatment with Dfcy was attenuated by U0126. Taken together, these results demonstrate that the iron chelator Dfcy exhibits anticancer effects via induction of ERK-dependent paraptosis.
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Quinasas MAP Reguladas por Señal Extracelular , Neoplasias , Humanos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Apoptosis , Quelantes del Hierro/farmacología , Línea Celular TumoralRESUMEN
Previous studies have revealed that age-related hearing loss (AHL) in Cdk5 regulatory subunit-associated protein 1 (Cdk5rap1)-knockout mice is associated with pathology in the cochlea. Here, we aimed to identify mitochondrial alterations in the cochlea of Cdk5rap1-knockout mice with AHL. Mitochondria in the spiral ganglion neurons (SGNs) and hair cells (HCs) were normal despite senescence; however, the mitochondria of types I, II, and IV spiral ligament fibrocytes were ballooned, damaged, and ballooned, respectively, in the stria vascularis. Our results suggest that the accumulation of dysfunctional mitochondria in the lateral wall, rather than the loss of HCs and SGNs, leads to the onset of AHL. Our results provide valuable information regarding the underlying mechanisms of AHL and the relationship between aberrant tRNA modification-induced hearing loss and mitochondrial dysfunction.
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Cóclea , Pérdida Auditiva , Animales , Ratones , Cóclea/metabolismo , Cóclea/patología , Pérdida Auditiva/genética , Pérdida Auditiva/metabolismo , Pérdida Auditiva/patología , Ratones Noqueados , Mitocondrias/genética , Mitocondrias/metabolismoRESUMEN
Although the core constituents of the Wnt/planar cell polarity (PCP) signaling have been extensively studied, their downstream molecules and protein-protein interactions have not yet been fully elucidated. Here, we show genetic and molecular evidence that the PCP factor, Vangl2, functionally interacts with the cell-cell adhesion molecule, N-cadherin (also known as Cdh2), for typical PCP-dependent neural development. Vangl2 and N-cadherin physically interact in the neural plates undergoing convergent extension. Unlike monogenic heterozygotes, digenic heterozygous mice with Vangl2 and Cdh2 mutants exhibited defects in neural tube closure and cochlear hair cell orientation. Despite this genetic interaction, neuroepithelial cells derived from the digenic heterozygotes did not show additive changes from the monogenic heterozygotes of Vangl2 in the RhoA-ROCK-Mypt1 and c-Jun N-terminal kinase (JNK)-Jun pathways of Wnt/PCP signaling. Thus, cooperation between Vangl2 and N-cadherin is at least partly via direct molecular interaction; it is essential for the planar polarized development of neural tissues but not significantly associated with RhoA or JNK pathways.
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Cadherinas , Tubo Neural , Ratones , Animales , Tubo Neural/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Polaridad Celular/genética , Vía de Señalización Wnt/fisiología , EpitelioRESUMEN
Ciliated cells in the airway epithelium generate mucus streams to remove extraneous particles and microorganisms by beating the motile cilia. This defense mechanism is crucial for maintaining homeostasis and preventing infection in the airway. Conventional methods to assess ciliary beating have revealed that rapid (>10 times per second) and metachronal beating of cilia enables efficient mucus transport. Cilia are oriented to excrete mucus toward the outside of the body. However, conventional methods to directly observe ciliary movements uses transmitted light, which requires translucent samples. Sliced or fragmented tissues are used to observe ciliary movements in thick human airway tissues. Therefore, conventional methods are unsuitable for assessing in situ orientation of ciliary movements. The orientation of ciliary beating can be indirectly analyzed by tracking particles spread onto the epithelium; however, the particles are not efficiently transported by immature cilia. To address this issue, we developed a method for labeling airway motile cilia with fluorescently labeled wheat germ agglutinin (FL-WGA). The new method enables microscopic observation of ciliary movements without slicing or fragmenting the airway tissues. Since the airway epithelium is observed from the apical side, in situ orientation of ciliary beating can be analyzed using this method. Additionally, epithelial damage, and the number and maturity of cilia can be assessed during the observation of ciliary beating. The new method, in combination with other methods, can provide more comprehensive data regarding ciliary movements.
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Cilios , Tráquea , Humanos , Epitelio , Moco , MovimientoRESUMEN
Hair follicles (HFs) undergo cyclic phases of growth, regression, and rest in association with hair shafts to maintain the hair coat. Nonsense mutations in the tight junction protein claudin (CLDN)-1 cause hair loss in humans. Therefore, we evaluated the roles of CLDNs in hair retention. Among the 27 CLDN family members, CLDN1, CLDN3, CLDN4, CLDN6, and CLDN7 were expressed in the inner bulge layer, isthmus, and sebaceous gland of murine HFs. Hair phenotypes were observed in Cldn1 weaker knockdown and Cldn3-knockout (Cldn1Δ/Δ Cldn3-/- ) mice. Although hair growth was normal, Cldn1Δ/Δ Cldn3-/- mice showed striking hair loss in the first telogen. Simultaneous deficiencies in CLDN1 and CLDN3 caused abnormalities in telogen HFs, such as an aberrantly layered architecture of epithelial cell sheets in bulges with multiple cell layers, mislocalization of bulges adjacent to sebaceous glands, and dilated hair canals. Along with the telogen HF abnormalities, which shortened the hair retention period, there was an enhanced proliferation of the epithelium surrounding HFs in Cldn1Δ/Δ Cldn3-/- mice, causing accelerated hair regrowth in adults. Our findings suggested that CLDN1 and CLDN3 may regulate hair retention in infant mice by maintaining the appropriate layered architecture of HFs, a deficiency of which can lead to alopecia.
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Alopecia , Animales , Ratones , Alopecia/genética , Claudina-1/genética , Claudina-1/metabolismo , Claudina-3/genética , Claudina-3/metabolismo , Claudina-4/metabolismo , Mutación , EnvejecimientoRESUMEN
Liquid-liquid phase separation (LLPS) is involved in various dynamic biological phenomena. In epithelial cells, dynamic regulation of junctional actin filaments tethered to the apical junctional complex (AJC) is critical for maintaining internal homeostasis against external perturbations; however, the role of LLPS in this process remains unknown. Here, after identifying a multifunctional actin nucleator, cordon bleu (Cobl), as an AJC-enriched microtubule-associated protein, we conducted comprehensive in vitro and in vivo analyses. We found that apical microtubules promoted LLPS of Cobl at the AJC, and Cobl actin assembly activity increased upon LLPS. Thus, microtubules spatiotemporally regulated junctional actin assembly for epithelial morphogenesis and paracellular barriers. Collectively, these findings established that LLPS of the actin nucleator Cobl mediated dynamic microtubule-actin cross-talk in junctions, which fine-tuned the epithelial barrier.
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Actinas , Proteínas de Microfilamentos , Actinas/metabolismo , Proteínas de Microfilamentos/metabolismo , Citoesqueleto de Actina/metabolismo , Uniones Intercelulares , Microtúbulos/metabolismoRESUMEN
Although sensorineural hearing loss (SHL) is relatively common, its cause has not been identified in most cases. Previous studies have suggested that viral infection is a major cause of SHL, especially sudden SHL, but the system that protects against pathogens in the inner ear, which is isolated by the blood-labyrinthine barrier, remains poorly understood. We recently showed that, as audiosensory receptor cells, cochlear hair cells (HCs) are protected by surrounding accessory supporting cells (SCs) and greater epithelial ridge (GER or Kölliker's organ) cells (GERCs) against viral infections. Here, we found that virus-infected SCs and GERCs induce HC death via production of the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). Notably, the HCs expressed the TRAIL death receptors (DR) DR4 and DR5, and virus-induced HC death was suppressed by TRAIL-neutralizing antibodies. TRAIL-induced HC death was not caused by apoptosis, and was inhibited by necroptosis inhibitors. Moreover, corticosteroids, the only effective drug for SHL, inhibited the virus-induced transformation of SCs and GERCs into macrophage-like cells and HC death, while macrophage depletion also inhibited virus-induced HC death. These results reveal a novel mechanism underlying virus-induced HC death in the cochlear sensory epithelium and suggest a possible target for preventing virus-induced SHL.
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Células Ciliadas Auditivas/virología , Pérdida Auditiva Sensorineural/virología , Necroptosis , Ligando Inductor de Apoptosis Relacionado con TNF/inmunología , Virosis/complicaciones , Animales , Células Cultivadas , Células Ciliadas Auditivas/inmunología , Células Ciliadas Auditivas/patología , Pérdida Auditiva Sensorineural/inmunología , Pérdida Auditiva Sensorineural/patología , Ratones Endogámicos ICR , Virosis/inmunología , Virosis/patologíaRESUMEN
Tissue-engineered tracheae have been developed to replace defective tracheae. However, the direction of ciliated cells in the regenerated epithelium remains unclear. We investigated planar polarity formed in the regenerated airway epithelium after tracheal graft implantation. We partially resected the rat trachea and implanted a collagen scaffold. The direction of the basal foot was assessed by transmission electron microscopy. Immunofluorescence staining was performed to examine the biased distribution of Vangl1 and Frizzled6 proteins. The direction of mucociliary transport was analyzed by video microscopy. Our results showed that the basal feet of cilia in the proximal and distal regions of the implanted areas were respectively oriented toward the proximal and distal directions. The biased distribution of Vangl1 and Frizzled6, and the directions of mucociliary transport showed that planar polarities formed in the regenerated epithelium were oriented toward the proximal, distal, left, and right directions in the proximal, distal, left, and right regions of the implanted area. These polarities persisted until nine months after implantation. Hence, the results suggest that planar polarities formed in epithelia regenerated on tracheal grafts are directed toward the nearby edges of implanted areas and are preserved for a prolonged period. The polarities can, at least partially, contribute to clearing external materials from the implanted areas by transporting them to a normal region.
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Cilios , Regeneración , Mucosa Respiratoria/citología , Ingeniería de Tejidos , Andamios del Tejido , Animales , Colágeno , Epitelio , Masculino , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Tráquea/trasplanteRESUMEN
Hearing loss has become one of the most common disabilities worldwide. The synaptic connections between inner hair cells (IHCs) and spiral ganglion neurons have specialized synaptic constructions, termed ribbon synapses, which are important for auditory function. The ribbon synapses in the cochlea are quite vulnerable to various insults. As such, the maintenance of ribbon synapses is important for ensuring hearing function. Insulin-like growth factor 1 (IGF1) plays a critical role in the development and maintenance of the cochlea and has the potential to protect cochlear hair cells from various insults. In this study, we examined the role of IGF1 in the maintenance of ribbon synapses in cochlear explants of postnatal day four mice. We cultured cochlear explants with an IGF1 receptor antagonist, JB1, which is an IGF1 peptide analog. Results showed that exposure to JB1 for 24 h resulted in the loss of ribbon synapses. After an additional 24-h culture without JB1, the number of ribbon synapses spontaneously recovered. The application of exogenous IGF1 showed two different aspects of ribbon synapses. Low doses of exogenous IGF1 promoted the recovery of ribbon synapses, while it compromised the spontaneous recovery of ribbon synapses at high doses. Altogether, these results indicate that the paracrine or autocrine release of IGF1 in the cochlea plays a crucial role in the maintenance of cochlear ribbon synapses.
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Multiciliated epithelial cells in the airway are essential for mucociliary clearance. Their function relies on coordinated, metachronal and directional ciliary beating, appropriate mucus secretion and airway surface hydration. However, current conventional methods for observing human airway ciliary movement require ciliated cells to be detached from airway tissues. Determining the directionality of cilia is difficult. We developed a novel method to stain airway epithelial cilia to observe their movement without releasing ciliated cells. Human tracheae were obtained from patients (n = 13) who underwent laryngectomies to treat malignancies or swallowing disorders. The tracheae were treated with fluorescently labeled wheat germ agglutinin, which interacts with the acidic mucopolysaccharides present on the cilia. Epithelial surfaces were observed using an epi-fluorescence microscope equipped with a water-immersion objective lens and a high-speed camera. Ciliary movement was observable at 125 fps (13/13 samples). Ciliated cells in close proximity mostly exhibited well-coordinated ciliary beats with similar directionalities. These findings indicated that wheat germ agglutinin renders ciliary beats visible, which is valuable for observing human airway ciliary movements in situ.
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Cilios/fisiología , Mucosa Respiratoria/citología , Coloración y Etiquetado/métodos , Tráquea/citología , Aglutininas del Germen de Trigo/química , Animales , Cilios/ultraestructura , Femenino , Colorantes Fluorescentes/química , Humanos , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Movimiento , Mucosa Respiratoria/fisiología , Tráquea/fisiologíaRESUMEN
To protect the audiosensory organ from tissue damage from the immune system, the inner ear is separated from the circulating immune system by the blood-labyrinth barrier, which was previously considered an immune-privileged site. Recent studies have shown that macrophages are distributed in the cochlea, especially in the spiral ligament, spiral ganglion, and stria vascularis; however, the direct pathogen defence mechanism used by audiosensory receptor hair cells (HCs) has remained obscure. Here, we show that HCs are protected from pathogens by surrounding accessory supporting cells (SCs) and greater epithelial ridge (GER or Kölliker's organ) cells (GERCs). In isolated murine cochlear sensory epithelium, we established Theiler's murine encephalomyelitis virus, which infected the SCs and GERCs, but very few HCs. The virus-infected SCs produced interferon (IFN)-α/ß, and the viruses efficiently infected the HCs in the IFN-α/ß receptor-null sensory epithelium. Interestingly, the virus-infected SCs and GERCs expressed macrophage marker proteins and were eliminated from the cell layer by cell detachment. Moreover, lipopolysaccharide induced phagocytosis of the SCs without cell detachment, and the SCs phagocytosed the bacteria. These results reveal that SCs function as macrophage-like cells, protect adjacent HCs from pathogens, and provide a novel anti-infection inner ear immune system.
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Células Ciliadas Auditivas Internas/fisiología , Células Ciliadas Auditivas Externas/fisiología , Células Laberínticas de Soporte/inmunología , Macrófagos/inmunología , Ganglio Espiral de la Cóclea/fisiología , Estría Vascular/fisiología , Animales , Animales Recién Nacidos , Escherichia coli/inmunología , Células Ciliadas Auditivas Internas/citología , Células Ciliadas Auditivas Externas/citología , Inmunidad Innata , Interferón-alfa/biosíntesis , Interferón-alfa/inmunología , Interferón beta/biosíntesis , Interferón beta/inmunología , Células Laberínticas de Soporte/citología , Células Laberínticas de Soporte/efectos de los fármacos , Células Laberínticas de Soporte/virología , Lipopolisacáridos/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/virología , Ratones , Ratones Endogámicos ICR , Técnicas de Cultivo de Órganos , Fagocitosis/efectos de los fármacos , Saccharomyces cerevisiae/inmunología , Ganglio Espiral de la Cóclea/citología , Estría Vascular/citología , Theilovirus/crecimiento & desarrollo , Theilovirus/patogenicidadRESUMEN
Enlarged vestibular aqueduct (EVA) is one of the most commonly identified inner ear malformations in hearing loss patients including Pendred syndrome. While biallelic mutations of the SLC26A4 gene, encoding pendrin, causes non-syndromic hearing loss with EVA or Pendred syndrome, a considerable number of patients appear to carry mono-allelic mutation. This suggests faulty pendrin regulatory machinery results in hearing loss. Here we identify EPHA2 as another causative gene of Pendred syndrome with SLC26A4. EphA2 forms a protein complex with pendrin controlling pendrin localization, which is disrupted in some pathogenic forms of pendrin. Moreover, point mutations leading to amino acid substitution in the EPHA2 gene are identified from patients bearing mono-allelic mutation of SLC26A4. Ephrin-B2 binds to EphA2 triggering internalization with pendrin inducing EphA2 autophosphorylation weakly. The identified EphA2 mutants attenuate ephrin-B2- but not ephrin-A1-induced EphA2 internalization with pendrin. Our results uncover an unexpected role of the Eph/ephrin system in epithelial function.
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Efrina-A2/genética , Bocio Nodular/genética , Pérdida Auditiva Sensorineural/genética , Transportadores de Sulfato/genética , Secuencia de Aminoácidos , Animales , Efrina-A1/genética , Efrina-A1/metabolismo , Efrina-A2/química , Efrina-A2/metabolismo , Efrina-B2/genética , Efrina-B2/metabolismo , Bocio Nodular/metabolismo , Pérdida Auditiva Sensorineural/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación Puntual , Unión Proteica , Receptor EphA2 , Transportadores de Sulfato/química , Transportadores de Sulfato/metabolismoRESUMEN
DNA polymerase δ, whose catalytic subunit is encoded by POLD1, is responsible for synthesizing the lagging strand of DNA. Single heterozygous POLD1 mutations in domains with polymerase and exonuclease activities have been reported to cause syndromic deafness as a part of multisystem metabolic disorder or predisposition to cancer. However, the phenotypes of diverse combinations of POLD1 genotypes have not been elucidated in humans. We found that five members of a multiplex family segregating autosomal recessive nonsyndromic sensorineural hearing loss (NS-SNHL) have revealed novel compound heterozygous POLD1 variants (p.Gly1100Arg and a presumptive null function variant, p.Ser197Hisfs*54). The recombinant p.Gly1100Arg polymerase δ showed a reduced polymerase activity by 30-40%, but exhibited normal exonuclease activity. The polymerase activity in cell extracts from the affected subject carrying the two POLD1 mutant alleles was about 33% of normal controls. We suggest that significantly decreased polymerase δ activity, but not a complete absence, with normal exonuclease activity could lead to NS-SNHL.
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ADN Polimerasa III/genética , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Variación Genética , Pérdida Auditiva/diagnóstico , Pérdida Auditiva/genética , Adulto , Alelos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Biomarcadores , ADN Polimerasa III/metabolismo , Activación Enzimática , Femenino , Estudios de Asociación Genética/métodos , Genotipo , Humanos , Masculino , Mutación , Linaje , Fenotipo , Polimorfismo de Nucleótido Simple , Hermanos , Síndrome , Secuenciación del ExomaRESUMEN
The tracheal lumen is essential for conducting air to the lung alveoli and for voice production. However, patients with severe tracheal stenosis and malignant tumors invading the trachea often require tracheal resection. Recently, various reported tissue engineering methods for tracheal reconstruction show that regeneration of ciliated epithelium in the reconstructed areas, as well as preservation of the luminal structure is possible. However, only few studies report on the mucociliary transport function in reconstructed tracheae. We investigated mucociliary transport function within rat tracheal epithelium, reorganized after autologous adipose tissue-derived stem cell (ASC) transplantation. Rat ASCs were expanded in culture, and then seeded in a collagen sponge, which was physically supported with a polypropylene framework. The ASC-seeded collagen sponge was transplanted into the rat tracheal defect. We then examined the motility and transport function of cilia generated in the transplanted area using ciliary beat frequency (CBF) and microsphere movement analyses. Our data suggested that autologous ASC transplantation promoted ciliogenesis, consistent with previous reports. The CBF analysis revealed that motility of the cilia generated in the ASC group was comparable to that observed in the normal rat tracheal epithelium. Transport function in the ASC group was higher than that in the control group. These data suggested that autologous ASC transplantation increased ciliated cells in the reconstructed area without significantly disrupting cilia motility, thereby promoting transport function regeneration. Autologous ASC transplantation is expected to be beneficial in morphological and functional regeneration of tracheal epithelium. Anat Rec, 303:471-477, 2020. © 2019 American Association for Anatomy.
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Cilios/fisiología , Colágeno/química , Células Madre/citología , Tráquea/fisiología , Tejido Adiposo/citología , Animales , Ratas , Ratas Sprague-Dawley , Regeneración , Ingeniería de Tejidos/métodos , Andamios del TejidoRESUMEN
TRIOBP remodels the cytoskeleton by forming unusually dense F-actin bundles and is implicated in human cancer, schizophrenia, and deafness. Mutations ablating human and mouse TRIOBP-4 and TRIOBP-5 isoforms are associated with profound deafness, as inner ear mechanosensory hair cells degenerate after stereocilia rootlets fail to develop. However, the mechanisms regulating formation of stereocilia rootlets by each TRIOBP isoform remain unknown. Using 3 new Triobp mouse models, we report that TRIOBP-5 is essential for thickening bundles of F-actin in rootlets, establishing their mature dimensions and for stiffening supporting cells of the auditory sensory epithelium. The coiled-coil domains of this isoform are required for reinforcement and maintenance of stereocilia rootlets. A loss of TRIOBP-5 in mouse results in dysmorphic rootlets that are abnormally thin in the cuticular plate but have increased widths and lengths within stereocilia cores, and causes progressive deafness recapitulating the human phenotype. Our study extends the current understanding of TRIOBP isoform-specific functions necessary for life-long hearing, with implications for insight into other TRIOBPopathies.