BUSCADOR RECOLECTA

The development of the inner ear complex cytoarchitecture and functional geometry requires the exquisite coordination of a variety of cellular processes in a temporal manner. At early stages of inner ear development several rounds of cell proliferation in the otocyst promote the growth of the structure. The apoptotic program is initiated in exceeding cells to adjust cell type numbers. Apoptotic cells are cleared by phagocytic cells that recognize the phosphatidylserine residues exposed in the cell membrane thanks to the energy supplied by autophagy. Specific molecular programs determine hair and supporting cell fate, these populations are responsible for the functions of the adult sensory organ: detection of sound, position and acceleration. The neurons that transmit auditory and balance information to the brain are also born at the otocyst by neurogenesis facilitated by autophagy. Cellular senescence participates in tissue repair, cancer and aging, situations in which cells enter a permanent cell cycle arrest and acquire a highly secretory phenotype that modulates their microenvironment. More recently, senescence has also been proposed to take place during vertebrate development in a limited number of transitory structures and organs; among the later, the endolymphatic duct in the inner ear. Here, we review these cellular processes during the early development of the inner ear, focusing on how the most recently described cellular senescence participates and cooperates with proliferation, apoptosis and autophagy to achieve otic morphogenesis and differentiation., This work has been supported by FP7-IAPP2013-TARGEAR to IVN, Spanish MINECO/FEDER SAF2017-86107-R to IVN and MM and SAF2015-65960-P to IP., Peer reviewed

Embryonic development requires the coordinated regulation of apoptosis, survival, autophagy, proliferation and differentiation programs. Senescence has recently joined the cellular processes required to master development, in addition to its well-described roles in cancer and ageing. Here, we show that senescent cells are present in a highly regulated temporal pattern in the developing vertebrate inner ear, first, surrounding the otic pore and, later, in the otocyst at the endolymphatic duct. Cellular senescence is associated with areas of increased apoptosis and reduced proliferation consistent with the induction of the process when the endolymphatic duct is being formed. Modulation of senescence disrupts otic vesicle morphology. Transforming growth factor beta (TGFβ) signaling interacts with signaling pathways elicited by insulin-like growth factor type 1 (IGF-1) to jointly coordinate cellular dynamics required for morphogenesis and differentiation. Taken together, these results show that senescence is a natural occurring process essential for early inner ear development., This work was supported by the European Commission FP7-PEOPLE-2013-IAPP TARGEAR to IVN and by the Spanish MINECO/FEDER SAF2017-86107-R to IVN and MM. SP was supported by a FPI predoctoral fellowship (BES-2015-071311) funded by MINECO/European Social Fund., Peer reviewed

Resumen del trabajo presentado al 42nd Congress of the Spanish Society of Biochemistry and Molecular Biology (SEBBM), celebrado en Madrid del 16 al 19 de julio de 2019., Sphingolipids are bioactive lipids whose role has progressed from being a mere component of cell membranes to be active participants in every cell biological process. Ceramide is the central backbone
precursor of all complex bioactive sphingolipids. A myriad of functions have been described for ceramide: cell death, cell differentiation, cell proliferation, senescence, autophagy, cell cycle arrest and
so on. We have addressed the study of the role that ceramide plays in the early development of inner ear in cultures of otic vesicles (OV), the primordium of this organ. This ex vivo culture mimics the in vivo development, maintaining the spatiotemporal pattern of morphogenesis. We had previously shown that a synthetic short-chain ceramide analogue was a potent inductor of apoptosis in OV cultures.
The action of ceramide is finished by its phosphorylation to ceramide-1-phosphate (C1P), a reaction catalysed by ceramide kinase (CERK). C1P has been reported to be cytoprotector in OV cultures. In the chicken embryo, the insulin-like growth factor -1 (IGF-1) is required for the survival and differentiation of epithelial auditory precursors. In this work we have studied whether the pro-survival role of IGF- 1 are due to the generation of C1P. To tackle the study we have used a specific inhibitor of CERK (NVP-231). Our results show that CERK is expressed during the early inner ear development in chicken. The inhibition of CERK decreased the OV size, reduced proliferation and increased cell cycle arrest followed by cell death. The inhibition of CERK also altered the neurogenesis in the acoustic
vestibular ganglion (AVG). Taken together, these results would confirm the involvement of C1P in the morphogenesis and maintenance of the OV and AVG. The exogenous IGF-1 treatment counteracted
partially the effect of the inhibitor, suggesting its protective role through CERK stimulation and C1P production., This work was supported by Spanish Ministerio de Economía, Industria y Competitividad (SAF2017-86107-RFEDER) to MM and IVN., Peer reviewed

Resumen del póster presentado al 42nd Congress of the Spanish Society of Biochemistry and Molecular Biology (SEBBM), celebrado en Madrid del 16 al 19 de julio de 2019., Ageing of the auditory system is associated with the incremental production of reactive oxygen species (ROS) and the accumulation of oxidative-derived damage in macromolecules, which contribute to cellular malfunction, compromise cell viability and, finally, causes functional decline. The cellular detoxification power partially relies in the production of the cofactor for the activity of key antioxidant enzymes NADPH. This is mainly produced by the glucose-6-phosphate dehydrogenase (G6PD), an enzyme that catalyzes the rate-limiting step in the pentose phosphate pathway. We show here that the transgenic mouse hG6PD-Tg, which shows enhanced NADPH production along life, maintains lower auditory thresholds than wild type mice during ageing. G6PD overexpression preserves irreplaceable cochlear cell populations, thus hG6PD-Tg mice exhibit significantly higher number of inner and outer hair cells (OHC) and more widespread OHC innervation than wild type mice. Transcripts for antioxidant enzymes were significantly increased in 3-month old hG6PD-Tg, particularly remarkable those of glutathione reductase and thioredoxin reductase 1, whose activity depends on NADPH availability. Accordingly, tyrosine modification by nitration in proteins was reduced in 9-month old G6PD-Tg compared with wild type mice. As well as lesser TUNEL positive apoptotic cells were detected in whole mount preparations of the organ of Corti of hG6PD-Tg mice. Western blotting analysis of cochlear extracts further showed increased survival and reduced pro-apoptotic signaling in the transgenic versus the wild type mice. In conclusion, we propose that NADPH overproduction from an early stage is an efficient mechanism to maintain the balance between the generation of ROS and the cell detoxification power along ageing and, therefore to prevent hearing loss progression., This work was supported by FP7-2013-TARGEAR and FEDER/SAF 2014-AGEAR and 2017-HEARCODE., Peer Reviewed

© 2019 Lassaletta, Calvino, Morales-Puebla, Lapunzina, Rodriguez-de la Rosa, Varela-Nieto and Martinez-Glez., Vestibular schwannomas (VSs) are benign tumors composed of differentiated neoplastic Schwann cells. They can be classified into two groups: sporadic VS and those associated with neurofibromatosis type 2 (NF2). VSs usually grow slowly, initially causing unilateral sensorineural hearing loss (HL) and tinnitus. These tumors cause HL both due to compression of the auditory nerve or the labyrinthine artery and due to the secretion of different substances potentially toxic to the inner ear or the cochlear nerve. As more and more patients are diagnosed and need to be managed, we are more than ever in need of searching for biomarkers associated with these tumors. Owing to an unknown toxic substance generated by the tumor, HL in VS may be linked to a high protein amount of perilymph. Previous studies have identified perilymph proteins correlated with tumor-associated HL, including μ-Crystallin (CRYM), low density lipoprotein receptor-related protein 2 (LRP2), immunoglobulin (Ig) γ-4 chain C region, Ig κ-chain C region, complement C3, and immunoglobulin heavy constant γ 3. Besides, the presence of specific subtypes of heat shock protein 70 has been suggested to be associated with preservation of residual hearing. It has been recently demonstrated that chemokine receptor-4 (CXCR4) is overexpressed in sporadic VS as well as in NF2 tumors and that hearing disability and CXCR4 expression may be correlated. Further, the genetic profile of VS and its relationship with poor hearing has also been studied, including DNA methylation, deregulated genes, growth factors, and NF2 gene mutations. The knowledge of biomarkers associated with VS would be of significant value to maximize outcomes of hearing preservation in these patients., This work was supported by a FEDER/Ministerio de Economía y Competitividad grant (SAF2017-86107-R) and by a Comunidad Autónoma de Madrid grant (B2017/BMD-3688).

Trabajo presentado en la XII Reunión Anual del CIBER de Enfermedades Raras, celebrada en San Lorenzo del Escorial (España) del 12 al 14 de marzo de 2019., La deficiencia en IGF-1 es una enfermedad rara (OMIM608747) que cursa con sordera neurosensorial y alteraciones neurológicas. El déficit de IGF-1 impide la correcta diferenciación neuronal en el ganglio auditivo del ratón y produce apoptosistemprana que progresa con la edad. Para facilitar el estudio de la deficiencia en IGF-1 y las alteraciones asociadas a la pérdida neuronal, se ha generado un modelo celular de la enfermedad humana en la línea de neuroblastoma murino Neuro2a mediante edición génica con CRISPR/Cas9. Dicho modelo reproduce la deleción parcial del exón 3 del gen Igf1 murino descrita como causante de pérdida auditiva en el hombre. Para la edición génica se transfectó el complejo crRNA:tracrRNA:Cas9 como ribonucleoproteína y se comprobó su incorporación en las células mediante microscopía detectando el tracrRNA marcado fluorescentemente. El ensayo de Surveyor detectó la presencia de mutaciones en las células, que se seleccionaron tras la clonación por dilución límite. La edición del gen Igf1 se comprobó por PCR en 56 clones y 5 de ellos se secuenciaron mediante el método Sanger. La secuenciación confirmó la deleción parcial de Igf1 en dos de los clones y se ha iniciado un estudio de su estado neuroinflamatorio, así como de su respuesta a distintos estímulos pro-inflamatorios, que puede contribuir a desvelar los mecanismos moleculares asociados a la deficiencia crónica de IGF-1., SAF2017 (86107-R); ACCI2016 (ER16P5AC7091); ACCI2017 (ER17P5AC7611/2017) y MouseAGE COST Action (BM1402)

[Introduction]: Excessive exposure to noise is a common occurrence that contributes to approximately 50% of the non-genetic hearing loss cases. Researchers need to develop standardized preclinical models and identify molecular targets to effectively develop prevention and curative therapies., [Areas covered]: In this review, the authors discuss the many facets of human noise-induced pathology, and the primary experimental models for studying the basic mechanisms of noise-induced damage, making connections and inferences among basic science studies, preclinical proofs of concept and clinical trials., [Expert opinion]: Whilst experimental research in animal models has helped to unravel the mechanisms of noise-induced hearing loss, there are often methodological variations and conflicting results between animal and human studies which make it difficult to integrate data and translate basic outcomes to clinical practice. Standardization of exposure paradigms and application of -omic technologies will contribute to improving the effectiveness of transferring newly gained knowledge to clinical practice., This work was supported by the grants Multi Target and View FEDER/CM-B2017/BMD-3688 from the Consejería de Educación e Investigación, Comunidad de Madrid and MINECO/FEDER SAF2017-86107-R from the Ministerio de Economía, Industria y Competitividad, Gobierno de España., Peer reviewed

Resumen del trabajo presentado en el 43rd Annual MidWinter Meeting-ARO, celebrado en San José, California (Estados Unidos) del 25 al 29 de enero de 2020., Ageing of the auditory system is associated with the incremental production of reactive oxygen species (ROS) and the accumulation of oxidative-derived damage in macromolecules, which contribute to cellular malfunction, compromise cell viability and, finally, causes functional decline. The cellular detoxification power partially relies in NADPH production, which is a cofactor for major cellular antioxidant enzymes. NADPH is mainly produced by glucose-6-phosphate dehydrogenase (G6PD), an enzyme that catalyzes the rate-limiting step in the pentose phosphate pathway. We show here that the transgenic mouse G6PD-Tg, which shows enhanced NADPH production along life, maintains lower auditory thresholds than wild type mice during ageing. G6PD overexpression preserved inner (IHC) and outer hair cells (OHC), OHC innervation and number of synapses per IHC. Transcripts for antioxidant enzymes were increased whereas levels of pro-apoptotic proteins were reduced in 3-month-old G6PD-Tg. Consequently, nitration of proteins, mitochondrial damage and TUNEL+ apoptotic cells were reduced in 9-month-old G6PDTg compared to wild type mice. Unexpectedly, G6PD overexpression triggered low grade inflammation that was effectively resolved in young mice, as shown by the absence of cochlear cellular damage and macrophages infiltration. In conclusion, we propose here that NADPH overproduction from an early stage is an efficient mechanism to maintain the balance between the generation of ROS and the cell detoxification power along ageing and, therefore to prevent hearing loss progression., This work has been founded by Spanish MINECO/ FEDER SAF2017-86107-R to IVN. JMBM was supported by contracts from MINECO/FEDER SAF2014-AGEAR and MINECO/FEDER SAF2017-86107-R.

Sphingolipids are bioactive lipid components of cell membranes with important signal transduction functions in health and disease. Ceramide is the central building block for sphingolipid biosynthesis and is processed to form structurally and functionally distinct sphingolipids. Ceramide can be phosphorylated by ceramide kinase (CERK) to generate ceramide-1-phosphate, a cytoprotective signaling molecule that has been widely studied in multiple tissues and organs, including the developing otocyst. However, little is known about ceramide kinase regulation during inner ear development. Using chicken otocysts, we show that genes for CERK and other enzymes of ceramide metabolism are expressed during the early stages of inner ear development and that CERK is developmentally regulated at the otic vesicle stage. To explore its role in inner ear morphogenesis, we blocked CERK activity in organotypic cultures of otic vesicles with a specific inhibitor. Inhibition of CERK activity impaired proliferation and promoted apoptosis of epithelial otic progenitors. CERK inhibition also compromised neurogenesis of the acoustic-vestibular ganglion. Insulin-like growth factor-1 (IGF-1) is a key factor for proliferation, survival and differentiation in the chicken otocyst. CERK inhibition decreased IGF-1-induced AKT phosphorylation and blocked IGF-1-induced cell survival. Overall, our data suggest that CERK is activated as a central element in the network of anti-apoptotic pro-survival pathways elicited by IGF-1 during early inner ear development., This work was supported by the Spanish Ministerio de Economia y Competitividad, project FEDER/SAF2017-86107-R to IVN and MM., Peer reviewed

Multi Target and View FEDER/CM-B2017/BMD-3688; MINECO/FEDER SAF2017-86107-R., zip file containing 6 folders Bioanalyzer folder; Figure 1 folder [Figure1_qPCR.xlsx; Figure1_ABR.xlsx; Figure1_ Amplitudes_SPSS.pdf; Figure1_Audiogram_SPSS.pdf; Figure1_ Latencies_SPSS.pdf; Figure1_MyeP0 IHQ base.txt; Figure1_MyeP0 IHQ middle.txt; Figure1_MyeP0 IHQ quantification.xlsx]; Figure 2 folder [Figure2_CtBP2GluR2/3_ whole mount_quantification.xlsx; Figure2_Hair cells counts whole mount_quantification.xlsx; Figure2_Neurofilament number of fibers_quantification; Figure2_Neurofilament Intensity OHC_quantification.xlsx]; Figure 3 folder [Figure3_Western Blot_quantification analysis.xlsx; Exp0_p22phox Mb2 20170608_1327 Prec8sectif_AllLanes.pdf; Exp0_PI3K Mb2 20170608_1500 Prec8sectif_AllLanes.pdf; Exp1_p22phox_Mb4_20180222_1340_Prec2sec_tapandotif_AllLanes.pdf; Exp1_PI3K_Mb4_20180216_1446_Prec4sectif_AllLanes.pdf; Figure3_3NT IHQ quantification intensity; Figure3_Cyt C Oxidase IHF quantification intensity.xlsx; Figure3_qPCR Source Data.xlsx]; Figure 4 folder [Figure4_Western Blot_quantification analysis.xlsx; Figure4_Western Blot_ImageQuant; Figure4_TUNEL+ whole mount_quantification.xlsx]; Figure 5 folder [Figure5_qPCR .xlsx; Figure5_IBA1 IHF quantification]., Peer reviewed

Animal models are invaluable for biomedical research, especially in the context of rare diseases, which have a very low prevalence and are often complex. Concretely mouse models provide key information on rare disease mechanisms and therapeutic strategies that cannot be obtained by using only alternative methods, and greatly contribute to accelerate the development of new therapeutic options for rare diseases. Despite this, the use of experimental animals remains controversial. The combination of respectful management, ethical laws and transparency regarding animal experimentation contributes to improve society's opinion about biomedical research and positively impacts on research quality, which eventually also benefits patients. Here we present examples of current advances in preclinical research in rare diseases using mouse models, together with our perspective on future directions and challenges.