RESUMEN
Protein homeostasis is essential to cell function, and a compromised ability to reduce the load of misfolded and aggregated proteins is linked to numerous age-related diseases, including hearing loss. Here, we show that altered proteostasis consequent to Elongator complex deficiency also impacts the proper development of the cochlea and results in deafness. In the absence of the catalytic subunit Elp3, differentiating spiral ganglion neurons display large aggresome-like structures and undergo apoptosis before birth. The cochlear mechanosensory cells are able to survive proteostasis disruption but suffer defects in polarity and stereociliary bundle morphogenesis. We demonstrate that protein aggregates accumulate at the apical surface of hair cells, where they cause a local slowdown of microtubular trafficking, altering the distribution of intrinsic polarity proteins and affecting kinocilium position and length. Alleviation of protein misfolding using the chemical chaperone 4-phenylbutyric acid during embryonic development ameliorates hair cell polarity in Elp3-deficient animals. Our study highlights the importance of developmental proteostasis in the cochlea and unveils an unexpected link between proteome integrity and polarized organization of cellular components.
Asunto(s)
Cóclea/citología , Cóclea/metabolismo , Células Ciliadas Auditivas/citología , Células Ciliadas Auditivas/fisiología , Proteostasis/fisiología , Polaridad Celular/genética , Polaridad Celular/fisiología , Técnica del Anticuerpo Fluorescente , Células HEK293 , Células Ciliadas Auditivas/metabolismo , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Humanos , Hibridación in Situ , Microscopía Confocal , Microscopía Electrónica de Rastreo , Modelos Biológicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Pliegue de Proteína , Proteostasis/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
Estrogen is the major female hormone involved in reproductive functions, but it also exerts a variety of additional roles in non-reproductive organs. In this review, we highlight the preclinical and clinical studies that have pointed out sex differences and estrogenic influence on audition. We also describe the experimental evidences supporting a protective role of estrogen towards acquired forms of hearing loss. Although a high level of endogenous estrogen is associated with a better hearing function, hormonal treatments at menopause have provided contradictory outcomes. The various factors that are likely to explain these discrepancies include the treatment regimen as well as the hormonal status and responsiveness of the patients. The complexity of estrogen signaling is being untangled and many downstream effectors of its genomic and non-genomic actions have been identified in other systems. Based on these advances and on the common physio-pathological events that underlie age-related, drug or noise-induced hearing loss, we discuss potential mechanisms for their protective actions in the cochlea.
Asunto(s)
Estrógenos/metabolismo , Audición , Animales , Cóclea/metabolismo , Cóclea/patología , Sordera/etiología , Sordera/metabolismo , Sordera/patología , Femenino , Humanos , Masculino , Receptores de Estrógenos/metabolismo , Caracteres Sexuales , Factores Sexuales , Transducción de SeñalRESUMEN
Hearing loss is a common affection mainly resulting from irreversible loss of the sensory hair cells of the cochlea; therefore, developing therapies to replace missing hair cells is essential. Understanding the mechanisms that drive their formation will not only help to unravel the molecular basis of deafness, but also give a roadmap for recapitulating hair cells development from cultured pluripotent stem cells. In this review, we provide an overview of the molecular mechanisms involved in hair cell production from both human and mouse embryonic stem cells. We then provide insights how this knowledge has been applied to differentiate induced pluripotent stem cells into otic progenitors and hair cells. Finally, we discuss the current limitations for properly obtaining functional hair cell in a Petri dish, as well as the difficulties that have to be overcome prior to consider stem cell therapy as a potential treatment for hearing loss.
Asunto(s)
Diferenciación Celular , Cóclea/citología , Células Ciliadas Auditivas/citología , Células Madre Pluripotentes/citología , Células Madre/citología , Animales , Pérdida Auditiva/terapia , Humanos , Ratones , Trasplante de Células Madre/métodosRESUMEN
PURPOSE: The aim of this study was to investigate the feasibility of tongue strength measures (TSMs) and the influence of bulb location, sex, and self-perceived pain and mucositis in head and neck cancer (HNC) patients during chemoradiotherapy (CRT). METHODS: Twenty-six newly diagnosed HNC patients treated with CRT performed anterior and posterior maximal isometric tongue pressures by means of the Iowa Oral Performance Instrument (IOPI). The Oral Mucositis Weekly Questionnaire (OMWQ) and a Visual Analogue Scale (VAS) for pain during swallowing were completed weekly from baseline to 1 week post CRT. RESULTS: Feasibility of TSMs during CRT declines significantly from 96 to 100% at baseline to 46% after 6 weeks of CRT. But post-hoc analyses reveal only significant differences in feasibility between baseline and measurements after 4 weeks of treatment. No effect of gender or bulb location was established, but feasibility is influenced by pain and mucositis. CONCLUSIONS: Feasibility of TSMs declines during CRT and is influenced by mucositis and pain. For the majority of subjects, TSMs were feasible within the first 4 weeks, which provides a window of scientific and clinical opportunities in this patient population.
Asunto(s)
Quimioradioterapia/efectos adversos , Trastornos de Deglución/etiología , Neoplasias de Cabeza y Cuello/radioterapia , Lengua/patología , Anciano , Trastornos de Deglución/patología , Femenino , Neoplasias de Cabeza y Cuello/patología , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Post-translational modifications (PTMs) are key molecular events that modify proteins after their synthesis and modulate their ultimate functional properties by affecting their stability, localisation, interaction potential or activity. These chemical changes expand the size of the proteome adding diversity to the molecular pathways governing the biological outcome of cells. PTMs are, thus, crucial in regulating a variety of cellular processes such as apoptosis, proliferation and differentiation and have been shown to be instrumental during embryonic development. In addition, alterations in protein PTMs have been implicated in the pathogenesis of many human diseases, including deafness. In this review, we summarize the recent progress made in understanding the roles of PTMs during cochlear development, with particular emphasis on the enzymes driving protein phosphorylation, acetylation, methylation, glycosylation, ubiquitination and SUMOylation. We also discuss how these enzymes may contribute to hearing impairment and deafness.
Asunto(s)
Sordera/patología , Audición/fisiología , Animales , Cóclea/crecimiento & desarrollo , Cóclea/metabolismo , Sordera/metabolismo , Histonas/metabolismo , Humanos , Presbiacusia/metabolismo , Presbiacusia/patología , Procesamiento Proteico-PostraduccionalRESUMEN
Lymphoid tyrosine phosphatase (LYP) and C-terminal Src kinase (CSK) are negative regulators of signaling mediated through the T-cell antigen receptor (TCR) and are thought to act in a cooperative manner when forming a complex. Here we studied the spatiotemporal dynamics of the LYP-CSK complex in T cells. We demonstrate that dissociation of this complex is necessary for recruitment of LYP to the plasma membrane, where it downmodulates TCR signaling. Development of a potent and selective chemical probe of LYP confirmed that LYP inhibits T-cell activation when removed from CSK. Our findings may explain the reduced TCR-mediated signaling associated with a single-nucleotide polymorphism that confers increased risk for certain autoimmune diseases, including type 1 diabetes and rheumatoid arthritis, and results in expression of a mutant LYP that is unable to bind CSK. Our compound also represents a starting point for the development of a LYP-based treatment of autoimmunity.
Asunto(s)
Activación de Linfocitos , Proteína Tirosina Fosfatasa no Receptora Tipo 22/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Linfocitos T/metabolismo , Proteína Tirosina Quinasa CSK , Membrana Celular/metabolismo , Regulación hacia Abajo , Humanos , Unión Proteica , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Familia-src QuinasasRESUMEN
The extracellular matrix (ECM) consists in a complex meshwork of collagens, glycoproteins, and proteoglycans, which serves a scaffolding function and provides viscoelastic properties to the tissues. ECM acts as a biomechanical support, and actively participates in cell signaling to induce tissular changes in response to environmental forces and soluble cues. Given the remarkable complexity of the inner ear architecture, its exquisite structure-function relationship, and the importance of vibration-induced stimulation of its sensory cells, ECM is instrumental to hearing. Many factors of the matrisome are involved in cochlea development, function and maintenance, as evidenced by the variety of ECM proteins associated with hereditary deafness. This review describes the structural and functional ECM components in the auditory organ and how they are modulated over time and following injury.
Asunto(s)
Sordera , Audición , Humanos , Audición/genética , Cóclea/metabolismo , Sordera/genética , Sordera/metabolismo , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismoRESUMEN
Retinoic acid receptors (RARs) heterodimerize with retinoid X receptors (RXRs) and bind to RA response elements (RAREs) in the regulatory regions of their target genes. Although previous studies on limited sets of RA-regulated genes have defined canonical RAREs as direct repeats of the consensus RGKTCA separated by 1, 2, or 5 nucleotides (DR1, DR2, DR5), we show that in mouse embryoid bodies or F9 embryonal carcinoma cells, RARs occupy a large repertoire of sites with DR0, DR8, and IR0 (inverted repeat 0) elements. Recombinant RAR-RXR binds these non-canonical spacings in vitro with comparable affinities to DR2 and DR5. Most DR8 elements comprise three half-sites with DR2 and DR0 spacings. This specific half-site organization constitutes a previously unrecognized but frequent signature of RAR binding elements. In functional assays, DR8 and IR0 elements act as independent RAREs, whereas DR0 does not. Our results reveal an unexpected diversity in the spacing and topology of binding elements for the RAR-RXR heterodimer. The differential ability of RAR-RXR bound to DR0 compared to DR2, DR5, and DR8 to mediate RA-dependent transcriptional activation indicates that half-site spacing allosterically regulates RAR function.
Asunto(s)
Receptores de Ácido Retinoico/metabolismo , Elementos de Respuesta , Receptores X Retinoide/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Unión Competitiva , Células Cultivadas , Inmunoprecipitación de Cromatina , Técnicas de Cocultivo , Secuencia de Consenso , Ensayo de Cambio de Movilidad Electroforética , Cuerpos Embrioides/metabolismo , Genoma , Ratones , Unión Proteica , Receptores de Ácido Retinoico/química , Secuencias Repetitivas de Ácidos Nucleicos , Receptores X Retinoide/química , Análisis de Secuencia de ADN , Volumetría , Transcripción GenéticaRESUMEN
It is widely accepted that cell fate determination in the cochlea is tightly controlled by different transcription factors (TFs) that remain to be fully defined. Here, we show that Sox9, initially expressed in the entire sensory epithelium of the cochlea, progressively disappears from differentiating hair cells (HCs) and is finally restricted to supporting cells (SCs). By performing ex vivo electroporation of E13.5-E14.5 cochleae, we demonstrate that maintenance of Sox9 expression in the progenitors committed to HC fate blocks their differentiation, even if co-expressed with Atoh1, a transcription factor necessary and sufficient to form HC. Sox9 inhibits Atoh1 transcriptional activity by upregulating Hey1 and HeyL antagonists, and genetic ablation of these genes induces extra HCs along the cochlea. Although Sox9 suppression from sensory progenitors ex vivo leads to a modest increase in the number of HCs, it is not sufficient in vivo to induce supernumerary HC production in an inducible Sox9 knockout model. Taken together, these data show that Sox9 is downregulated from nascent HCs to allow the unfolding of their differentiation program. This may be critical for future strategies to promote fully mature HC formation in regeneration approaches.
Asunto(s)
Cóclea , Células Ciliadas Auditivas , Epitelio , Diferenciación Celular , ElectroporaciónRESUMEN
Post-translational modifications of proteins are essential for the proper development and function of many tissues and organs, including the inner ear. Ubiquitination is a highly selective post-translational modification that involves the covalent conjugation of ubiquitin to a substrate protein. The most common outcome of protein ubiquitination is degradation by the ubiquitin-proteasome system (UPS), preventing the accumulation of misfolded, damaged, and excess proteins. In addition to proteasomal degradation, ubiquitination regulates other cellular processes, such as transcription, translation, endocytosis, receptor activity, and subcellular localization. All of these processes are essential for cochlear development and maintenance, as several studies link impairment of UPS with altered cochlear development and hearing loss. In this review, we provide insight into the well-oiled machinery of UPS with a focus on its confirmed role in normal hearing and deafness and potential therapeutic strategies to prevent and treat UPS-associated hearing loss.
Asunto(s)
Sordera , Ubiquitina , Humanos , Ubiquitina/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitinación , AudiciónRESUMEN
BACKGROUND: Radiation-associated-dysphagia is a serious side effect of radiotherapy (RT) for head and neck cancer (HNC). METHODS: Seventy-six patients had a weekly prospective follow-up from baseline until one week post-RT. Combined mixed model analysis (n = 43) determined the evolution of self-perceived swallowing function, isometric tongue strength (MIP), tongue strength (TS) during swallowing (Pswal), and quality of life (QoL) in these patients during RT. RESULTS: Swallowing deteriorated from the third week on, resulting in an increase of tube dependency from 10% at baseline toward 31% post-RT. Both MIP and Pswal are reduced, with anterior MIP decreasing in 29% of patients and posterior MIP in 17%. Pswal decreases for saliva and a bolus swallow. All QoL subscales except "sleep" were affected during RT. CONCLUSIONS: Self-perceived swallowing function, TS and QoL decrease during RT for HNC. Current findings highlight the need for early monitoring of these parameters.
Asunto(s)
Trastornos de Deglución/etiología , Neoplasias de Cabeza y Cuello/radioterapia , Calidad de Vida , Radioterapia/efectos adversos , Lengua/efectos de la radiación , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Estudios de Seguimiento , Fuerza de la Mano , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Escala Visual AnalógicaRESUMEN
INTRODUCTION: Overexpression of the ERBB2 oncogene is observed in about 20% of human breast tumors and is the consequence of increased transcription rates frequently associated with gene amplification. Several studies have shown a link between activator protein 2 (AP-2) transcription factors and ERBB2 gene expression in breast cancer cell lines. Moreover, the Yin Yang 1 (YY1) transcription factor has been shown to stimulate AP-2 transcriptional activity on the ERBB2 promoter in vitro. In this report, we examined the relationships between ERBB2, AP-2alpha, and YY1 both in breast cancer tissue specimens and in a mammary cancer cell line. METHODS: ERBB2, AP-2alpha, and YY1 protein levels were analyzed by immunohistochemistry in a panel of 55 primary breast tumors. ERBB2 gene amplification status was determined by fluorescent in situ hybridization. Correlations were evaluated by a chi2 test at a p value of less than 0.05. The functional role of AP-2alpha and YY1 on ERBB2 gene expression was analyzed by small interfering RNA (siRNA) transfection in the BT-474 mammary cancer cell line followed by real-time reverse transcription-polymerase chain reaction and Western blotting. RESULTS: We observed a statistically significant correlation between ERBB2 and AP-2alpha levels in the tumors (p < 0.01). Moreover, associations were found between ERBB2 protein level and the combined high expression of AP-2alpha and YY1 (p < 0.02) as well as between the expression of AP-2alpha and YY1 (p < 0.001). Furthermore, the levels of both AP-2alpha and YY1 proteins were inversely correlated to ERBB2 gene amplification status in the tumors (p < 0.01). Transfection of siRNAs targeting AP-2alpha and AP-2gamma mRNAs in the BT-474 breast cancer cell line repressed the expression of the endogenous ERBB2 gene at both the mRNA and protein levels. Moreover, the additional transfection of an siRNA directed against the YY1 transcript further reduced the ERBB2 protein level, suggesting that AP-2 and YY1 transcription factors cooperate to stimulate the transcription of the ERBB2 gene. CONCLUSION: This study highlights the role of both AP-2alpha and YY1 transcription factors in ERBB2 oncogene overexpression in breast tumors. Our results also suggest that high ERBB2 expression may result either from gene amplification or from increased transcription factor levels.
Asunto(s)
Neoplasias de la Mama/metabolismo , Genes erbB-2/genética , Factor de Transcripción AP-2/biosíntesis , Factor de Transcripción YY1/biosíntesis , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/genética , Línea Celular Tumoral , Femenino , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Factor de Transcripción AP-2/genética , Factor de Transcripción YY1/genéticaRESUMEN
The Hedgehog (Hh) signaling pathway plays an important role in human development. Abnormal activation of this pathway has been observed in several types of human cancers, such as the upper gastro-intestinal tract cancers. However, activation of the Hh pathway in colorectal cancers is controversial. We analyzed the expression of the main key members of the Hh pathway in 7 colon cancer cell lines in order to discover whether the pathway is constitutively active in these cells. We estimated the expression of SHH, IHH, PTCH, SMO, GLI1, GLI2, GLI3, SUFU and HHIP genes by RT-PCR. Moreover, Hh ligand, Gli3 and Sufu protein levels were quantified by western blotting. None of the cell lines expressed the complete set of Hh pathway members. The ligands were absent from Colo320 and HCT116 cells, Smo from Colo205, HT29 and WiDr. GLI1 gene was not expressed in SW480 cells nor were GLI2/GLI3 in Colo205 or Caco-2 cells. Furthermore the repressive form of Gli3, characteristic of an inactive pathway, was detected in SW480 and Colo320 cells. Finally treatment of colon cancer cells with cyclopamine, a specific inhibitor of the Hh pathway, did not downregulate PTCH and GLI1 genes expression in the colorectal cells, whereas it did so in PANC1 control cells. Taken together, these results indicate that the aberrant activation of the Hh signaling pathway is not common in colorectal cancer cell lines.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias Colorrectales/metabolismo , Proteínas Hedgehog/metabolismo , Receptores de Superficie Celular/genética , Transducción de Señal , Factores de Transcripción/genética , Alcaloides de Veratrum/farmacología , Western Blotting , Línea Celular Tumoral , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Regulación hacia Abajo/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Receptores Patched , Receptor Patched-1 , Receptores de Superficie Celular/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Tomatina/análogos & derivados , Tomatina/farmacología , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Proteína con Dedos de Zinc GLI1RESUMEN
MicroRNAs are important regulators of gene expression and are involved in cellular processes such as proliferation or differentiation, particularly during development of numerous organs including the inner ear. However, it remains unknown if miRNAs are required during the earliest stages of otocyst and cochlear duct development. Here, we report that a conditional loss of Dicer expression in the otocyst impairs the early development of the inner ear as a result of the accumulation of DNA damage that trigger p53-mediated apoptosis. Moreover, cochlear progenitors in the prosensory domain do not exit the cell cycle. Our unbiased approach identified ItgA3 as a target of miR-183, which are both enriched in the otic vesicle. We observed that the repression of integrin alpha 3 by miR-183 controls cell proliferation in the developing cochlea. Collectively, our results reveal that Dicer and miRNAs play essential roles in the regulation of early inner ear development.
Asunto(s)
Oído Interno/embriología , Integrina alfa3/fisiología , MicroARNs/fisiología , Animales , Diferenciación Celular/fisiología , Línea Celular , Cóclea/citología , Cóclea/embriología , ARN Helicasas DEAD-box/genética , Femenino , Ratones , Ratones Noqueados , Embarazo , Ribonucleasa III/genética , Transducción de SeñalRESUMEN
The oncoprotein HER-2/neu is a prosurvival factor, and its overexpression has been correlated with poor prognosis in patients with breast cancer. We report that HER-2 is a new substrate for caspase-8 and that tumor necrosis factor alpha (TNF-alpha) stimulation leads to an early caspase-8-dependent HER-2 cleavage in MCF7 A/Z breast adenocarcinoma cells defective for nuclear factor kappaB (NFkappaB) activation. We show that the antiapoptotic transcription factor NFkappaB counteracts this cleavage through induction of the caspase-8 inhibitor c-FLIP. Our results also demonstrate that this HER-2 cleavage contributes to the TNF-alpha-induced apoptosis pathway because ectopic expression of an uncleavable HER-2 protects NFkappaB-defective cells against TNF-alpha-mediated cell death. Therefore, we propose an original model in which NFkappaB exerts a new antiapoptotic function by counteracting TNF-alpha-triggered cleavage of the HER-2 survival factor.
Asunto(s)
Adenocarcinoma/metabolismo , Neoplasias de la Mama/metabolismo , Proteínas Portadoras/metabolismo , Caspasas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , FN-kappa B/metabolismo , Receptor ErbB-2/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Adenocarcinoma/enzimología , Adenocarcinoma/patología , Antiinfecciosos/farmacología , Apoptosis/fisiología , Sitios de Unión , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Caspasa 8 , Inhibidores de Caspasas , Línea Celular Tumoral , Cicloheximida/farmacología , Activación Enzimática , Humanos , FN-kappa B/antagonistas & inhibidores , Nitrilos , Receptor ErbB-2/biosíntesis , Receptor ErbB-2/genética , Estimulación Química , Sulfonas , Activación TranscripcionalRESUMEN
Overexpression of the ERBB2 gene occurs in 30% of human breast cancers and is correlated with poor prognosis. The deregulation is the consequence of an increased transcription level and gene amplification. Several laboratories, including our own, have identified, in the proximal promoter, enhancers implicated in the gene overexpression. However, our previous studies of a 6-kb ERBB2 promoter fragment revealed the presence of repressing fragments, which were able to overcome the effect of the proximal enhancers. These repressing elements were functional in all cell lines, regardless of their endogenous ERBB2 expression level. Here, we show that a distal ERBB2 promoter region restores high transcription rates specifically in ERBB2 overexpressing breast cancer cells. This distal promoter region thus contains enhancers essential for the overexpression of the gene. By EMSA, performed with nuclear extract of cells overexpressing (BT-474) or not (MDA-MB-231) the ERBB2 gene, we show that at least two sequences of the distal promoter region are bound exclusively by BT-474 extract. Further experiments reveal that AP-2 transcription factors contribute to this differential binding activity, by binding recognition sequences located 4500 bp and 4000 bp upstream of the transcription start site. These sites are occupied by AP2 in vivo, as demonstrated by ChIP assay. Inactivation of AP-2 proteins in ERBB2 overexpressing cells reduces the distal promoter activity up to 70%, indicating the AP-2 factors are implicated in the strong distal enhancing effect. Moreover, we identified a 54-bp fragment that is bound specifically by BT-474 nuclear extract. Further experiments did not lead to the identification of the protein responsible for this binding. Our results thus highlight the importance of ERBB2 distal promoter region and further implicate AP-2 in ERBB2 overexpression in breast cancer cells.
Asunto(s)
Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica , Genes erbB-2/genética , Regiones Promotoras Genéticas/genética , Receptor ErbB-2/metabolismo , Secuencia de Bases , Neoplasias de la Mama/genética , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Ensayo de Cambio de Movilidad Electroforética , Vectores Genéticos , Humanos , Datos de Secuencia Molecular , Oligonucleótidos , Factor de Transcripción AP-2/metabolismoRESUMEN
Sound signal is detected by sensory hair cells located in the cochlear region of the inner ear, and transmitted to the central nervous system by the spiral ganglion neurons (SGNs). These bipolar neurons develop long peripheral processes to connect hair cells, forming ribbon synapses, specialised for the precision and speed required to process auditory information. The establishment of a complex innervation pattern relies on specific signals, intrinsic to SGNs or provided by neighbouring cells, which are tightly controlled in time and space. In this paper, we review recent advances about stepwise development of afferent auditory neuronal circuitries, from neuron specification within the early otic vesicle to definitive synaptic connections with target cells. We especially focus on the cellular and molecular developmental changes involved in fibre outgrowth and extension to the sensory epithelium, specific afferent targeting to hair cells, and synaptic pruning.
Asunto(s)
Cóclea/inervación , Nervio Coclear/fisiología , Células Ciliadas Auditivas/fisiología , Audición , Neurogénesis , Transmisión Sináptica , Animales , Vías Auditivas/fisiología , Percepción Auditiva , Cóclea/crecimiento & desarrollo , Nervio Coclear/crecimiento & desarrollo , Nervio Coclear/metabolismo , Células Ciliadas Auditivas/metabolismo , Humanos , Morfogénesis , Proteínas del Tejido Nervioso/metabolismo , Ganglio Espiral de la Cóclea/crecimiento & desarrolloRESUMEN
The organ of Corti, the auditory organ of the mammalian inner ear, contains sensory hair cells and supporting cells that arise from a common sensory progenitor. The molecular bases allowing the specification of these progenitors remain elusive. In the present study, by combining microarray analyses with conditional deletion of Dicer in the developing inner ear, we identified that miR-124 controls cell fate in the developing organ of Corti. By targeting secreted frizzled-related protein 4 (Sfrp4) and Sfrp5, two inhibitors of the Wnt pathway, we showed that miR-124 controls the ß-catenin-dependent and also the PCP-related non-canonical Wnt pathways that contribute to HC differentiation and polarization in the organ of Corti. Thus, our work emphasizes the importance of miR-124 as an epigenetic safeguard that fine-tunes the expression of genes critical for cell patterning during cochlear differentiation.
Asunto(s)
Células Ciliadas Auditivas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Células Laberínticas de Soporte/metabolismo , MicroARNs/genética , Proteínas Proto-Oncogénicas/genética , beta Catenina/genética , Regiones no Traducidas 3' , Proteínas Adaptadoras Transductoras de Señales , Animales , Secuencia de Bases , Diferenciación Celular/genética , Polaridad Celular , ARN Helicasas DEAD-box/deficiencia , ARN Helicasas DEAD-box/genética , Embrión de Mamíferos , Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Células Ciliadas Auditivas/citología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Células Laberínticas de Soporte/citología , Ratones , MicroARNs/metabolismo , Datos de Secuencia Molecular , Organogénesis/genética , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ribonucleasa III/deficiencia , Ribonucleasa III/genética , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismoRESUMEN
The importance of tyrosine phosphorylation in normal cell physiology is well established, highlighted by the many human diseases that stem from abnormalities in protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) function. Contrary to earlier assumptions, it is now clear that both PTKs and PTPs are highly specific, non-redundant, and tightly regulated enzymes. Hematopoietic cells express particularly high numbers of PTKs and PTPs, and aberrant function of these proteins have been linked to many hematopoietic disorders. While PTK inhibitors are among FDA approved drugs for the treatment of leukemia and other cancers, efforts to develop therapeutics that target specific PTPs are still in its infancy. Here, we describe methods on how to evaluate effects of PTP inhibitors on T cell receptor signaling. Moreover, we provide a comprehensive strategy for compound prioritization, applicable to any drug discovery project involving T cells. We present a testing funnel that starts with relatively high-throughput luciferase reporter assays, followed by immunoblot, calcium flux, flow cytometry, and proliferation assays, continues with cytokine bead arrays, and finishes with specificity assays that involve RNA interference. We provide protocols for experiments in the Jurkat T cell line, but more importantly give detailed instructions, paired with numerous tips, on how to prepare and work with primary human T cells.
Asunto(s)
Descubrimiento de Drogas/métodos , Inhibidores Enzimáticos/farmacología , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/efectos de los fármacos , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Humanos , Células Jurkat , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño , Transducción de Señal/efectos de los fármacos , Linfocitos T/enzimologíaRESUMEN
Cord blood hematopoietic progenitor cells (CB-HPCs) transplanted immunodeficient NOD/LtsZ-scidIL2Rγ(null) (NSG) and NOD/SCID/IL2Rγ(null) (NOG) mice need efficient human cell engraftment for long-term HIV-1 replication studies. Total body irradiation (TBI) is a classical myeloablation regimen used to improve engraftment levels of human cells in these humanized mice. Some recent reports suggest the use of busulfan as a myeloablation regimen to transplant HPCs in neonatal and adult NSG mice. In the present study, we further ameliorated the busulfan myeloablation regimen with fresh CB-CD34+cell transplantation in 3-4 week old NSG mice. In this CB-CD34+transplanted NSG mice engraftment efficiency of human CD45+cell is over 90% in peripheral blood. Optimal engraftment promoted early and increased CD3+T cell levels, with better lymphoid tissue development and prolonged human cell chimerism over 300 days. These humanized NSG mice have shown long-lasting viremia after HIV-1JRCSF and HIV-1Bal inoculation through intravenous and rectal routes. We also saw a gradual decline of the CD4+T cell count, widespread immune activation, up-regulation of inflammation marker and microbial translocation after HIV-1 infection. Humanized NSG mice reconstituted according to our new protocol produced, moderate cellular and humoral immune responses to HIV-1 postinfection. We believe that NSG mice reconstituted according to our easy to use protocol will provide a better in vivo model for HIV-1 replication and anti-HIV-1 therapy trials.