A network of growth and transcription factors controls neuronal differentation and survival in the developing ear

Inner ear neurons develop from the otic placode and connect hair cells with central neurons in auditory brain stem nuclei. Otic neurogenesis is a developmental process which can be separated into different cellular states that are characterized by a distinct combination of molecular markers. Neurogenesis is highly regulated by a network of extrinsic and intrinsic factors, whose participation in auditory neurogenesis is discussed. Trophic factors include the fibroblast growth factor, neurotrophins and insulin-like peptide families. The expression domains of transcription factor families and their roles in the regulation of intracellular signaling pathways associated with neurogenesis are also discussed. Understanding and defining the key factors and gene networks in the development and function of the inner ear represents an important step towards defeating deafness. © UBC Press.This work has been supported in part by grants from the Spanish Ministries of Health and Education (PI0-5168 and BFU-200500084), from the Community of Madrid (CAM-PRICIT0530), the Royal Society, DIGNA Biotech and Mutua Madrileña. Hortensia Sánchez-Calderón holds a postdoctoral I3P CSIC- Fondo Social Europeo contract.Peer Reviewe

AKT Signaling Mediates IGF-I Survival Actions on Otic Neural Progenitors

Background: Otic neurons and sensory cells derive from common progenitors whose transition into mature cells requires the coordination of cell survival, proliferation and differentiation programmes. Neurotrophic support and survival of post-mitotic otic neurons have been intensively studied, but the bases underlying the regulation of programmed cell death in immature proliferative otic neuroblasts remains poorly understood. The protein kinase AKT acts as a node, playing a critical role in controlling cell survival and cell cycle progression. AKT is activated by trophic factors, including insulin-like growth factor I (IGF-I), through the generation of the lipidic second messenger phosphatidylinositol 3-phosphate by phosphatidylinositol 3-kinase (PI3K). Here we have investigated the role of IGF-dependent activation of the PI3K-AKT pathway in maintenance of otic neuroblasts. Methodology/Principal Findings: By using a combination of organotypic cultures of chicken (Gallus gallus) otic vesicles and acoustic-vestibular ganglia, Western blotting, immunohistochemistry and in situ hybridization, we show that IGF-I-activation of AKT protects neural progenitors from programmed cell death. IGF-I maintains otic neuroblasts in an undifferentiated and proliferative state, which is characterised by the upregulation of the forkhead box M1 (FoxM1) transcription factor. By contrast, our results indicate that post-mitotic p27Kip-positive neurons become IGF-I independent as they extend their neuronal processes. Neurons gradually reduce their expression of the Igf1r, while they increase that of the neurotrophin receptor, TrkC. Conclusions/Significance: Proliferative otic neuroblasts are dependent on the activation of the PI3K-AKT pathway by IGF-I for survival during the otic neuronal progenitor phase of early inner ear development

RAF Kinase Activity Regulates Neuroepithelial Cell Proliferation and Neuronal Progenitor Cell Differentiation during Early Inner Ear Development

Background: Early inner ear development requires the strict regulation of cell proliferation, survival, migration and differentiation, coordinated by the concerted action of extrinsic and intrinsic factors. Deregulation of these processes is associated with embryonic malformations and deafness. We have shown that insulin-like growth factor I (IGF-I) plays a key role in embryonic and postnatal otic development by triggering the activation of intracellular lipid and protein kinases. RAF kinases are serine/threonine kinases that regulate the highly conserved RAS-RAF-MEK-ERK signaling cascade involved in transducing the signals from extracellular growth factors to the nucleus. However, the regulation of RAF kinase activity by growth factors during development is complex and still not fully understood. Methodology/Principal Findings: By using a combination of qRT-PCR, Western blotting, immunohistochemistry and in situ hybridization, we show that C-RAF and B-RAF are expressed during the early development of the chicken inner ear in specific spatiotemporal patterns. Moreover, later in development B-RAF expression is associated to hair cells in the sensory patches. Experiments in ex vivo cultures of otic vesicle explants demonstrate that the influence of IGF-I on proliferation but not survival depends on RAF kinase activating the MEK-ERK phosphorylation cascade. With the specific RAF inhibitor Sorafenib, we show that blocking RAF activity in organotypic cultures increases apoptosis and diminishes the rate of cell proliferation in the otic epithelia, as well as severely impairing neurogenesis of the acoustic-vestibular ganglion (AVG) and neuron maturation. Conclusions/Significance: We conclude that RAF kinase activity is essential to establish the balance between cell proliferation and death in neuroepithelial otic precursors, and for otic neuron differentiation and axonal growth at the AVG

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

Early otic development depends on autophagy for apoptotic cell clearance and neural differentiation

Autophagy is a highly regulated program of self-degradation of the cytosolic constituents that has key roles during early development and in adult cell growth and homeostasis. To investigate the role of autophagy in otic neurogenesis, we studied the expression of autophagy genes in early stages of chicken (Gallus gallus) inner ear development and the consequences of inhibiting the autophagic pathway in organotypic cultures of explanted chicken otic vesicles (OVs). Here we show the expression of autophagy-related genes (Atg) Beclin-1 (Atg6), Atg5 and LC3B (Atg8) in the otocyst and the presence of autophagic vesicles by using transmission electron microscopy in the otic neurogenic zone. The inhibition of the transcription of LC3B by using antisense morpholinos and of class III phosphatidylinositol 3-kinase with 3-methyladenine causes an aberrant morphology of the OV with accumulation of apoptotic cells. Moreover, inhibition of autophagy provokes the misregulation of the cell cycle in the otic epithelium, impaired neurogenesis and poor axonal outgrowth. Finally, our results indicate that autophagy provides the energy required for the clearing of neuroepithelial dying cells and suggest that it is required for the migration of otic neuronal precursors. Taken together, our results show for the first time that autophagy is an active and essential process during early inner ear development. © 2012 Macmillan Publishers Limited. All rights reserved.Peer Reviewe

Autofagia en el desarrollo temprano del oído interno

Resumen del póster presentado al XXXIV Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Barcelona del 5 al 8 de septiembre de 2011.La autofagia es una vía lisosomal de degradación muy conservada que previene la acumulación de proteínas y orgánulos dañados y que tiene un papel clave en el desarrollo de vertebrados. El desarrollo temprano del oído interno requiere una estricta regulación de los procesos de proliferación celular, supervivencia, migración y diferenciación con el fin de generar la mayoría de los tipos celulares del oído interno adulto incluyendo las neuronas del ganglio auditivo-vestibular. Para investigar el papel de la autofagia en el desarrollo temprano del oído interno se ha examinado la expresión de la maquinaria molecular de la autofagia y las consecuencias de su inhibición con 3-metiladenina (3-MA) en cultivos organotípicos de explantes de vesícula ótica. En este trabajo demostramos que el mecanismo de autofagia está presente en el oído interno en desarrollo in vivo y que los inhibidores de la autofagia modifican la relación LC3-I/LC3II. El bloqueo de la autofagia con 3-MA causa la acumulación de células TUNEL-positivas y la desregulación del ciclo celular. Además, la neurogénesis ótica se ve gravemente afectada tanto en la producción del número adecuado de precursores neuronales como en el correcto crecimiento axonal. Finalmente, nuestros resultados indican que la autofagia proporciona la energía necesaria para que se produzca la fagocitosis de las células neuroepiteliales que mueren por apoptosis. En conjunto, nuestros resultados demuestran que la autofagia es un proceso activo durante el desarrollo temprano del oído interno, siendo esencial para la morfogénesis del epitelio ótico y la organización del ganglio auditivo-vestibular.Peer Reviewe

Comparative gene expression study of the vestibular organ of the Igf1 deficient mouse using whole-transcript arrays

The auditory and vestibular organs form the inner ear and have a common developmental origin. Insulin like growth factor 1 (IGF-1) has a central role in the development of the cochlea and maintenance of hearing. Its deficiency causes sensorineural hearing loss in man and mice. During chicken early development, IGF-1 modulates neurogenesis of the cochleovestibular ganglion but no further studies have been conducted to explore the potential role of IGF-1 in the vestibular system. In this study we have compared the whole transcriptome of the vestibular organ from wild type and Igf1-/- mice at different developmental and postnatal times. RNA was prepared from E18.5, P15 and P90 vestibular organs of Igf1-/- and Igf1+/+ mice and the transcriptome analysed in triplicates using Affymetrix® Mouse Gene 1.1 ST Array Plates. These plates are whole-transcript arrays that include probes to measure both messenger (mRNA) and long intergenic non-coding RNA transcripts (lincRNA), with a coverage of over 28 thousand coding transcripts and over 7 thousands non-coding transcripts. Given the complexity of the data we used two different methods VSN-RMA and mmBGX to analyse and compare the data. This is to better evaluate the number of false positives and to quantify uncertainty of low signals. We identified a number of differentially expressed genes that we described using functional analysis and validated using RT-qPCR. The morphology of the vestibular organ did not show differences between genotypes and no evident alterations were observed in the vestibular sensory areas of the null mice. However, well-defined cellular alterations were found in the vestibular neurons with respect their number and size. Although these mice did not show a dramatic vestibular phenotype, we conducted a functional analysis on differentially expressed genes between genotypes and across time. This was with the aim to identify new pathways that are involved in the development of the vestibular organ as well as pathways that maybe affected by the lack of IGF-1 and be associated to the morphological changes of the vestibular neurons that we observed in the Igf1-/- mice.This study was supported by grants from FP7-PEOPLE-2013-IAPP 612261-TARGEAR to IVN, and CIBERER-INTRA/09/761.2 to IVN and JD. We thank Begoña Rodriguez for expert technical assistance with the celloidin material. LR-dlR and SM-C hold CIBERER contracts.Peer Reviewe

Autophagy is required for apoptotic cell clearance and neural differentiation in early otic development

Resumen del póster presentado al 50th Inner Ear Biology Workshop, celebrado en Alcala de Henares-Madrid (España) del 10 al 13 de septiembre de 2013.Autophagy is a highly regulated program of self-degradation of the cytosolic constituents that has key roles during early development and in adult cell growth and homeostasis. To investigate the role of autophagy in otic neurogenesis, we studied the expression of autophagy genes in early stages of chicken inner ear development and the consequences of inhibiting the autophagic pathway in organotypic cultures of explanted chicken otic vesicles. Here we show the expression of autophagy-related genes Beclin-1, Atg5 and LC3B during early development of the chicken inner ear. The otic epithelium shows intense lysosomal activity and numerous autophagic vesicles, especially at the neuroblasts exit zone.The inhibition of the transcription of LC3B by using both genetic and pharmacological approaches causes an aberrant morphology of the otic vesicle with accumulation of apoptotic cells. Moreover, inhibition of autophagy provokes the misregulation of the cell cycle in the otic epithelium, impaired neurogenesis and poor axonal outgrowth. Finally, the addition of methyl pyruvate abrogated the consequences of autophagy inhibition. Therefore, our results indicate that autophagy provides the energy required for the clearing of neuroepithelial dying cells and suggest that it is required for the migration of otic neuronal precursors. Taken together, our results show for the first time that autophagy is an active and essential process during early inner ear development.This work was supported in part by the Instituto de Salud Carlos II, Centro de Investigación en Red en Enfermedades Raras CIBERER, and MICINN (SAF2008-00470).Peer Reviewe

Neurotrophic action of insulin-like growth factor-I in the inner ear

6 páginas, 4 figuras.-- et al.[ES]: [Introducción]: La pérdida de audición constituye una de las deficiencias sensoriales invalidantes más frecuentes en el mundo desarrollado. En la actualidad se estudian diferentes abordajes terapéuticos, entre los que se incluyen el tratamiento con células madre, la manipulación genética y la protección farmacológica. [Objetivo]: Evaluar el papel del factor de crecimiento similar a la insulina de tipo I (IGF-I) en el desarrollo, el mantenimiento y la reparación de la función auditiva. [Desarrollo]: El desarrollo del oído interno depende de la adecuada coordinación de los procesos celulares de proliferación, diferenciación, neurogénesis y muerte celular programada, que se encuentran regulados por distintos factores entre los que se encuentra el IGF-I. Durante la embriogénesis del oído interno, este factor se expresa abundantemente y es fundamental para la supervivencia celular y el mantenimiento de los precursores neuronales. El estudio del ratón nulo Igf-1¿/¿ ha puesto de manifiesto su importancia en el desarrollo y mantenimiento funcional del oído interno. Los ratones deficientes en este gen presentan alteraciones morfológicas que se corresponden con graves deficiencias funcionales, confirmadas mediante el análisis de los potenciales evocados auditivos de tronco cerebral. El déficit de IGF-I en humanos también se acompaña de hipoacusia sensorial profunda. [Conclusión]: En este escenario, se perfila el IGF-I como un factor clave para el desarrollo de la función auditiva y un candidato para la terapia regenerativa del oído interno.[EN]: [Introduction]: Loss of hearing constitutes one of the most frequent disabling sensory impairments in the developed world. Different therapeutic approaches are currently being studied, including treatment with stem cells, genetic manipulation and pharmacological protection. [Aim]: To evaluate the role played by insulin-like growth factor-I (IGF-I) in the development, maintenance and repair of auditory functioning. [Development]: Proper development of the inner ear is dependent on a suitable coordination of the cell processes of proliferation, differentiation, neurogenesis and programmed cell death, which are regulated by different factors, one of which is IGF-I. During the embryogenesis of the inner ear, this factor is expressed in abundance and is essential for cell survival and maintaining neuronal precursors. Studies conducted in Igf-1–/– null mice have highlighted its importance in the development and continued functioning of the inner ear. Mice with a deficit in this gene display morphological disorders that correspond to severe functional deficiencies, which are confirmed by analysing brainstem auditory evoked potentials. A deficit of IGF-I in humans is also accompanied by profound sensory hypoacusis. [Conclusions]: In a scenario like this, IGF-I appears as a key factor in the development of auditory functioning and a candidate for regenerative therapy of the inner ear.Trabajo financiado en parte gracias a los siguientes proyectos de investigación: FIS03/203, BMC03-07751, PIO05, BFU05, CAM IV PRICIT, y a la colaboración de la empresa DIGNA Biotech. H. Sánchez-Calderón disfruta de un contrato posdoctoral I3P del CSIC.Peer Reviewe