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

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., Hearing loss is the most common sensory deficit in the human population. Mutations in the gene coding for IGF-1 cause sensorineural hearing loss in man and mice. Actions of IGF-1 are mediated
by binding to its high affinity transmembrane receptor, IGF1R. This interaction typically leads to the activation of the PI3K-AKT pathway and of the MAPK-ERK cascade. To gain insight into the molecular mechanisms involved in IGF-1 downstream signaling in the sensory hair cells, we have used HEI-OC1 (House Ear Institute-Organ of Corti 1), a cell line derived from the auditory organ of the transgenic ImmortomouseTMat postnatal day 7. The study of the relative expression of genes of the IGF system by RT-qPCR showed that IGF system factors and receptors are expressed in HEI-OC1 and that Igf1 transcripts are more abundant than those of Igf2 and Ins1/ 2. We also studied the cellular actions
and downstream signaling pathways of IGF-1. Cell cycle and apoptosis were studied by flow cytometry and immunohistochemistry, whilst activation of target proteins was measured by Western blotting. The consequences of blockage of IGF-1 actions were analyzed by using specific IGF1R inhibitors and XTT assay. IGF-1 increased survival, proliferation, as well as glucose metabolism and protein synthesis, whereas autophagic flux was decreased and apoptosis inhibited. In conclusion, HEI-OC1 cells can be used as a model to understand the actions of IGF-1 in hair cells, to identify novel targets and to unravel the molecular mechanisms involved in IGF-1 deficiency-associated otic damage., This work was supported by the Spanish Ministerio de Economía y Competitividad (MINECO)/FEDER
SAF2017-86107-R. AGM holds a MECD FPU16/03308 fellowship., Peer reviewed

Resumen del trabajo presentado al 42st Annual Association for Research in Otolaryngology (ARO) MidWinter Meeting, celebrado en Baltimore, Maryland (USA) del 9 al 13 de febrero de 2019., Nutritional imbalance is an emerging causative factor for deafness and several epidemiological and experimental studies have linked alterations in methionine metabolism caused by folic acid and/or vitamin B12 deficiencies with age-related, noiseinduced or sudden hearing loss. Betaine homocysteine S-methyltransferases (BHMTs) are methionine cycle enzymes that remethylate homocysteine, hence their malfunction leads to hyperhomocysteinemia. Here, we have studied the expression of methionine cycle genes in the mouse cochlea and the impact of knocking out the Bhmt gene in the auditory receptor. We evaluated age-related changes in mouse hearing by recording auditory brainstem responses at different moments from embryonic to adult stages, and following exposure to noise. Also we measured cochlear cytoarchitecture, gene expression by RNA-arrays and RT-qPCR, and metabolite levels in liver and plasma by HPLC. Our results indicate that there is an age-dependent strain-specific expression of methionine cycle genes in the mouse cochlea and a further regulation during the response to noise damage. Loss of Bhmt did not cause an evident impact in the hearing acuity of young mice, but it produced higher threshold shifts and poorer
recovery following noise challenge. Hearing loss was associated with increased cochlear injury, altered expression of cochlear methionine cycle genes and hyperhomocysteinemia. Our results suggest that BHMT plays a central role in the homeostasis of cochlear methionine metabolism and that Bhmt2 upregulation could carry out a compensatory role in cochlear protection against noise injury in the absence of BHMT., Work supported by the Spanish MINECO/FEDER SAF2017-86107-R grant to IVN, U.S. National Institutes of Health DK056350 to SZ, and CEU-Banco Santander precompetitive (MUSPB047) and consolidation (MBS18C12) project to TP., Peer Reviewed

Resumen del trabajo presentado al 42st Annual Association for Research in Otolaryngology (ARO) MidWinter Meeting, celebrado en Baltimore, Maryland (USA) del 9 al 13 de febrero de 2019., P38 mitogen-activated protein (MAP) kinases p38α, β, γ and δ are codified by Mapk14, 11, 12 and 13 genes, respectively. P38 kinases are activated by phosphorylation in response to a variety of extracellular stress stimuli that transduce to the nuclei. The MAPK14 (p38a) isoform is ubiquitously expressed and is critical for mammalian embryonic development. MAPK14 participates in the cochlear response to stressors like cisplatin, UV radiation or noise exposure. MAPK14 expression and activity also increase as a consequence of Igf1 deletion in mice in association with hearing loss
and cellular apoptosis. We have studied the expression pattern of the four p38 isoforms along ageing in wild type and knock out Igf1 null mice, to show that they are age- and IGF-1- regulated. To further explore the role of Mapk14 in the cochlea, we have studied the Mapk14lox/loxRERTnERT/ERT conditional knock out mouse and cell lines derived from embryonic fibroblasts. Mapk14 knock out mice auditory brainstem responses (ABR) recording showed that ABR thresholds do not vary upon Mapk14 deletion induced by tamoxifen treatment. RT-qPCR gene expression studies of the p38 isoforms indicated that other isoforms are induced and maybe taking over MAPK14 cochlear functions. P38 isoforms relative expression levels also varied in Mapk14-knock out fibroblasts, as well as the transcripts for MEF2, FoxM1 and FoxG1, which are also altered in the cochlea of Igf1-null mice. Finally, we assessed the response to noise-induced injury in Cre/loxP Mapk14 knock out mice before and after noise exposure. Interestingly, mice with total or partial deletion of Mapk14 exposed to violet noise at an intensity of 105 dB SPL during 30 minutes showed a better recovery of hearing thresholds than wild type mice. These data suggest that MAPK14 plays a central role in the progression of cochlear damage after noise exposure., Research was supported by Spanish MINECO/FEDER SAF2017-86107-R and EU FP7-PEOPLE-TARGEAR grants to IV-N. LRR and SMC hold CIBERER contracts. AGM holds a MECD FPU fellowship., Peer Reviewed

Resumen del póster presentado al 6th Symposium on Biomedical Research: Advances and Perspectives in Molecular Endocrinology "In Homage to Gabriella Morreale", celebrado en el Instituto de Investigaciones Biomédicas Alberto Sols (IIBM-CSIC) el 31 de mayo de 2019., World Health Organization estimates that around 466 million people worldwide have disabling hearing loss (HL), and 34 million of these are children. The most common is sensorineural hearing loss (SNHL), a heterogeneous disorder which is produced mainly by the irreversible loss of sensory cells or neurons in the cochlea. Insulin like growth factor type 1 (IGF-1) is a neurotrophic factor key for the regulation of postnatal cochlear growth and differentiation. Human IGF-1 deficiency is a rare disease (ORPHA73272) associated with growth retardation, microcephaly and SNHL. The mouse model lacking the Igf1 gene reproduces the syndrome and presents dwarfism and SNHL. IGF-1 deficiency causes important cellular alterations in the mouse cochlea, such as the early apoptosis of auditory neurons and the deficit in myelination. Analysis of downstream signalling in the Igf1-/- cochlea has shown the activation of p38 MAPK pathway, involved in response to stress, whereas ERK1/2 and AKT pathways, which regulate proliferation and survival, are impaired. A transcriptomic study carried out in the Igf1-/- showed the altered expression of the cell cycle modulator Foxm1 and of the myocyte enhancer factor-2 (Mef2), a key factor for cellular differentiation, during inner ear development and early postnatal ages.IGF-1 haploinsufficiency has been also associated with growth retardation and HL in human genetic disorders such as Laron syndrome. In contrast, the Igf1+/- mouse does not show congenital HL, but adult mice hearing thresholds progressively increase with ageing and mice show increased susceptibility to noise insult. Adult Igf1+/- cochleae show unbalanced redox and inflammation biomarkers, as well as altered IGF-1 downstream signalling, which have been proposed as molecular mechanism underlying susceptibility. Mouse models of IGF-1 deficiency constitute a valuable tool to study the molecular bases of deafness and to identify potential targets to develop new HL therapies., This work was supported by grants from the Spanish MINECO/FEDER (SAF2014-53979-R and SAF2017-86107-R) and FP7-PEOPLE-2013-IAPP TARGEAR to IVN. SMC and LRdR holds a contract supported by CIBERER (Institute of Health Carlos III) co-financed with FEDER funds., Peer Reviewed

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)

Trabajo presentado en el 56th Inner Ear Biology Workshop, celebrado en Padua (Italia) del 7 al 10 de septiembre de 2019., In all vertebrates, the inner ear originates from the otic placode, an ectodermal patch in the head which invaginates and closes to form the otic vesicle (OV) or otocyst. The OV is considered the anlagen of the inner ear since their epithelial cells will differentiate generating most of the cell types building the complex structure of the adult inner ear. This transition is carried out by spatio-temporal restricted processes of proliferation, differentiation, migration, apoptosis, autophagy and senescence. A network of intracellular signals manages the precise process of development. The knowledge of the molecular bases that guide otic development is necessary for the design of novel treatments for the protection and repair of cells in hearing loss disorders.
One of the emerging family of compounds with critical roles in most cell biological processes are the bioactive sphingolipids. In fact the term ¿morphogenetic lipids¿ are coined for sphingolipids that regulate stem cells survival and differentiation during embryonic and postnatal development. Ceramide is the central backbone precursor of all complex bioactive sphingolipids. 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 the ceramide kinase (CERK). The presence of CERK has not been studied in the OV.
In the chicken embryo, Insulin-like growth factor-1 (IGF-1) is required for the survival and differentiation of the epithelial auditory precursors. In addition, IGF-1 deficit causes syndromic deafness in mice and men. 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 CERK inhibitor (NVP-231). We have addressed the study in ex vivo cultures of OVs described as a good model of inner ear development that mimics the in vivo program maintaining the morphogenetic spatiotemporal pattern.
Our results show that CERK is expressed during the developing inner ear in chicken. The inhibition of CERK 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 of the OV and AVG. The inhibitor counteracted the effect of IGF-1 exogenously added, suggesting that a part of the IGF-1 protective role could be carried out by CERK stimulation and C1P production., This work was supported by Spanish Ministerio de Economía, Industria y Competitividad (SAF2017-86107-R-FEDER) to MM and IVN.

Trabajo presentado en el 6th Symposium on Biomedical Research "Advances and Perspectives in Molecular Endocrinology", celebrado en Madrid (España) el 31 de mayo de 2019., Insulin-like growth factor-1 (IGF-1) is a key element in chicken otic development, and its deficit causes syndromic deafness in mice and men (1). Early steps of inner ear development involve the formation of an autonomous embryonic structure, the otic vesicle or otocyst. Otic vesicle development requires the coordinated actions of apoptosis, proliferation, senescence, cell differentiation and autophagy (2). Autophagy is a catabolic process essential for vertebrate development and homeostasis. We have reported that autophagy is required to facilitate the clearance of apoptotic cells and neuronal differentiation during early otic development (3). In other cellular context, IGF-1 has been shown to regulate autophagy (4). To better understand the molecular actions of IGF-1 and its potential modulation of autophagy in otic cells, we have used the cell line HEI-OC1 (derived from the auditory organ of the transgenic mouse Immortomouse¿) and ex vivo cultures of chicken otocysts. IGF-1 signaling pathway was studied by Western blotting of main IGF-1 targets. Proliferation was measured by incorporation of EdU. Apoptosis was assessed by TUNEL labelling and flow cytometry. Autophagy flux was analyzed by measuring LC3 and p62 relative protein levels. Our results show that IGF-1 promotes cell survival and cell proliferation while reduces cell death in both experimental models. IGF-1 is also playing a role in neuronal differentiation in the otic vesicle, maintaining otic neuroblasts in an undifferentiated and proliferative state. Furthermore IGF-1 actions include the downregulation of autophagy, which is induced in response to starvation and is essential for development and differentiation of otic cells., This work was supported by FEDER/SAF2017-86107-R-HEARCODE. SP holds an FPI predoctoral fellowship (BES-2015- 071311; European Social Fund/MINECO). AGM holds a MECD FPU fellowship (FPU16/03308). LR-R holds a contract supported by CIBERER (Institute of Health Carlos III) co-financed with FEDER funds

Resumen del trabajo presentado al 43rd Annual MidWinter Meeting-ARO, celebrado en San Jose, California (US) del 25 al 29 de enero de 2020., 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 ., Peer reviewed

Resumen del trabajo presentado al 43rd Annual MidWinter Meeting-ARO, celebrado en San Jose, California (US) del 25 al 29 de enero de 2020., This work was supported by FEDER/SAF2017-86107-R-HEARCODE. SP holds an FPI predoctoral fellowship (BES-2015-071311; European Social Fund/MINECO). AGM holds a MECD FPU fellowship (FPU16/03308). LR-R holds a contract supported by CIBERER (Institute of Health Carlos III) co-financed with FEDER funds., 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.