© 2021 by the authors., Insulin-like growth factor 1 (IGF-1) is a peptide hormone belonging to the insulin family of proteins. Almost all of the biological effects of IGF-1 are mediated through binding to its high-affinity tyrosine kinase receptor (IGF1R), a transmembrane receptor belonging to the insulin receptor family. Factors, receptors and IGF-binding proteins form the IGF system, which has multiple roles in mammalian development, adult tissue homeostasis, and aging. Consequently, mutations in genes of the IGF system, including downstream intracellular targets, underlie multiple common pathologies and are associated with multiple rare human diseases. Here we review the contribution of the IGF system to our understanding of the molecular and genetic basis of human hearing loss by describing, (i) the expression patterns of the IGF system in the mammalian inner ear; (ii) downstream signaling of IGF-1 in the hearing organ; (iii) mouse mutations in the IGF system, including upstream regulators and downstream targets of IGF-1 that inform cochlear pathophysiology; and (iv) human mutations in these genes causing hearing loss., This research was funded by Spanish FEDER/CM, B2017/BMD-3688; FEDER/MICIN, PID2020-115274RB-I00-THEARPY and EU H2020-INTERREG, 0551_PSL_6_E grants to I.V.-N. and ACCI/ISCIII, ER19P5AC761 grant to L.R.-d.l.R. Á.G.-M. holds a FPU (FPU16/03308; MECD) contract. S.M.-C. and L.R.-d.l.R. hold CIBER ISCIII researcher contracts.

Insulin-like growth factor 1 (IGF-1) is a trophic factor for the nervous system where it exerts pleiotropic effects, including the regulation of metabolic homeostasis. IGF-1 deficiency induces morphological alterations in the cochlea, apoptosis and hearing loss. While multiple studies have addressed the role of IGF-1 in hearing protection, its potential function in the modulation of otic metabolism remains unclear. Here, we report that “House Ear Institute-organ of Corti 1” (HEI-OC1) auditory cells express IGF-system genes that are regulated during their differentiation. Upon binding to its high-affinity receptor IGF1R, IGF-1 activates AKT and mTOR signaling to stimulate anabolism and, concomitantly, to reduce autophagic catabolism in HEI-OC1 progenitor cells. Notably, IGF-1 stimulation during HEI-OC1 differentiation to mature otic cells sustained both constructive metabolism and autophagic flux, possibly to favor cell remodeling. IGF1R engagement and downstream AKT signaling promoted HEI-OC1 cell survival by maintaining redox balance, even when cells were challenged with the ototoxic agent cisplatin. Our findings establish that IGF-1 not only serves an important function in otic metabolic homeostasis but also activates antioxidant defense mechanisms to promote hair cell survival during the stress response to insults., MCIN/AEI/10.13039/ 501100011033 THEARPY-PID2020-115274RB-I00; 0551_PSL_6_E POCTEP FGCSIC/ PSL-INTERREG/FEDER NITROPROHEAR, zip file containing 7 folders: Figure 2 folder [Figure 2_Blots Report.pdf; Figure_2C_qPCR.xlsx; Figure_2D_qPCR.xlsx; Figure_2E&G_Data&Analysis.xlsx; Figure_2F_Data&Analysis.xlsx] Figure 3 folder [Figure 3_Blots Report.pdf; Figure_3B_Data&Analysis.xlsx; Figure_3C_Data&Analysis.xlsx] Figure 4 folder [Figure 4_Blots Report.pdf;Figure_4A_Data&Analysis.xlsx; Figure_4B_Data&Analysis.xlsx; Figure_4C&D_Data&Analysis.xlsx] Figure 5 folder [Figure 5_Blots Report.pdf; Figure_5A_Data&Analysis.xlsx; Figure_5B_Data&Analysis.xlsx; Figure_5C-D_Data&Analysis.xlsx; Figure_5E-F_Data&Analysis.xlsx] Figure 6 folder [Figure 6_Blots Report.pdf; Figure_6B_Data&Analysis.xlsx; Figure_6C_Data&Analysis.xlsx; Figure_6D_Data&Analysis.xlsx] Figure 7 folder [Figure 7_Blots Report.pdf; Figure_7B_Oxyblot_Data&Analysis.xlsx; Figure_7B_qPCR_Data&Analysis.xlsx; Figure_7B_WesternBlotting_Data&Analysis.xlsx; Figure_7C_Data&Analysis.xlsx; Figure_7D_Data&Analysis.xlsx; Figure_7E_Data&Analysis.xlsx] Supplementary Material folder [Figure_S2_Data&Analysis.xlsx], Peer reviewed

The current Research Topic tries to highlight some of the most relevant recent advances regarding sensorineural hearing loss (SNHL). The etiology of SNHL primarily involves hereditary factors, ototoxic drugs, noise, and aging, all of which trigger two main mechanisms: damage to the organ of Corti, namely mechanosensory hair cells (HCs) and non-sensory supporting cells (SCs), and/or loss of spiral ganglion neurons (SGNs) that subsequently form the auditory nerve and hence connect the inner ear to the brain., TJ was supported by the NIH/NIDCD (K08DC019683). IV-N was supported by PID2020-115274RB-I00 from the Spanish MCIN/AEI/10.13039/501100011033 and FEDER.

Cellular senescence is a stable anti-proliferative state, which has an essential role in cell balance control in diverse physiological and pathological settings (Chan and Narita, 2019; Gorgoulis et al., 2019). Senescence research is a highly dynamic field that has experienced a radical expansion over the last few years with the identification of the role of senescence in a growing list of diseases and physiological processes and the promise for therapeutic interventions based on senescence (Munoz-Espin and Serrano, 2014; Paez-Ribes et al., 2019). The current Research Topic aims to give an overview of the latest advances in this field highlighting the progress in understanding the mechanism of senescence and its link to disease in the nervous system and other organs. The issue includes a wide range of articles, including original research reports, mini-reviews, and reviews that explore diverse angles of this topic, showcasing the current trends in senescence research., Research in the authors’ laboratories was supported by the following grants: RTI2018-098520-B-I00 from the Spanish MCIN/AEI/10.13039/501100011033 and FEDER to IP. 2020ΣE01300001 from the Ministry of Development and Investment, 775 (Hippo) and 3782 (PACOREL) from the Hellenic Foundation for Research and Innovation, 70/3/8916 from NKUA-SARG, the Welfare Foundation for Social & Cultural Sciences (KIKPE), and H. Pappas donation to VG, PID2020-115274RB-I00 from the Spanish MCIN/AEI/10.13039/501100011033 and FEDER to IV-N. IP and IV-N were members of the Spanish Senescence Network, Senestherapy (RED2018-102698-T).

Trabajo presentado en el Spanish Symposium on IGFs and Insulin 2022: Implications in Physiology and Disease, celebrado en Logroño (España) del 21 al 22 de abril de 2022., Human deficiency of insulin-like growth factor type 1 (IGF-1) causes a rare disorder (OMIM608747; ORPHA73272), which leads to sensorineural hearing loss and neurological disorders [1,2]. The Igf1-deficient mouse replicates this neurological phenotype syndrome, and shows impaired neuronal differentiation and increased apoptosis of auditory neurons [3,4]. IGF-1 is a hormone associated with decreased neuroinflammation [5] and increased cellular senescence, though molecular mechanisms involved are poorly understood [6]. In order to study IGF-1 deficiency and understand the alterations linked to neuronal loss, a cellular model of the human disease was generated in the murine neuroblastoma cell line N2a by using CRISPR/Cas9 technology. The crRNA:tracrRNA:Cas9 complex was transfected as a ribonucleoprotein and clones were isolated by limiting dilution. Sanger and next-generation sequencing confirmed Igf1 gene editing. Clones 4A10 and 2G3, with a mutation frequency above 90%, were selected for the study. Both clones, which differentially expressed IGF system factors and proinflammatory cytokines, showed alterations in bleomycin-induced senescence and increased resistance to cisplatin treatment. Chronic IGF-1 deficiency leads to changes in the response to ototoxic drugs such as cisplatin and bleomycin in neuroblastoma cells. These cell lines are an opportunity to unravel new molecular mechanisms of neuronal damage associated with chronic IGF1 deficiency., THEARPY-PID2020-115274RB-I00 funded by MCIN/AEI/10.13039/501100011033 and NITROPROHEAR (0551_PSL_6_E POCTEP FGCSIC/PSL-INTERREG/FEDER).

Insulin-like growth factor 1 (IGF-1) is a trophic factor for the nervous system where it exerts pleiotropic effects, including the regulation of metabolic homeostasis. IGF-1 deficiency induces morphological alterations in the cochlea, apoptosis and hearing loss. While multiple studies have addressed the role of IGF-1 in hearing protection, its potential function in the modulation of otic metabolism remains unclear. Here, we report that “House Ear Institute-organ of Corti 1” (HEI-OC1) auditory cells express IGF-system genes that are regulated during their differentiation. Upon binding to its high-affinity receptor IGF1R, IGF-1 activates AKT and mTOR signaling to stimulate anabolism and, concomitantly, to reduce autophagic catabolism in HEI-OC1 progenitor cells. Notably, IGF-1 stimulation during HEI-OC1 differentiation to mature otic cells sustained both constructive metabolism and autophagic flux, possibly to favor cell remodeling. IGF1R engagement and downstream AKT signaling promoted HEI-OC1 cell survival by maintaining redox balance, even when cells were challenged with the ototoxic agent cisplatin. Our findings establish that IGF-1 not only serves an important function in otic metabolic homeostasis but also activates antioxidant defense mechanisms to promote hair cell survival during the stress response to insults., This research was funded by Spanish MCIN/AEI/10.13039/501100011033 THEARPYPID2020-115274RB-I00; 0551_PSL_6_E POCTEP FGCSIC/ PSL-INTERREG/FEDER NITROPROHEAR and CA20121 COST Action/EU—BenBedPhar grants to I.V.-N. Á.G.-M. holds an FPU (FPU16/03308; MECD) fellowship, and L.R.-d.l.R. holds a CIBER ISCIII researcher contract., Peer reviewed

[Background] The blood-labyrinth barrier (BLB) separates the inner ear from the circulation and is critical for maintaining ionic homeostasis and limiting the entry of deleterious agents. BLB integrity is disrupted by bacterial lipopolysaccharide (LPS), which elicits a strong inflammatory response in the inner ear leading to irreversible otic damage. Prolonged administration of systemic corticosteroids is the available treatment, but it shows both limited efficacy and major adverse effects. SPT-2101 is a novel in situ-forming gel formulation of dexamethasone allowing slow and sustained drug release after single intratympanic administration., [Methods] We used a rat model of LPS-induced injury to define the functional, cellular and molecular mechanisms associated to BLB dysfunction and the protection by SPT-2101. Hearing was assessed by auditory brainstem response (ABR) recording, BLB permeability by gadolinium dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and Evans blue extravasation. Gross cochlear histology and cellular alterations were studied by hematoxylin-eosin staining and immunofluorescence. RT-qPCR, PCR array and western blotting were used to assess transcriptional and protein changes., [Results] LPS-challenged rats showed BLB breakdown and altered permeability as shown by the progressive increase in cochlear gadolinium uptake and Evans blue incorporation. LPS administration increased the cochlear expression of the LPS toll-like receptors Tlr2 and co-receptor Cd14, pro-inflammatory cytokines and receptors such as Il1b and ll1r1, and also the oxidative stress and inflammasome mediators NRF2 and NLRP3. LPS also increased IBA1-positive macrophage infiltration in the lateral wall and spiral ganglion. A single intratympanic injection of SPT-2101 protected BLB integrity and prevented otic injury. Comparable effects were obtained by repeated administration of systemic dexamethasone, but not by a single dose. SPT-2101 administration normalized molecular inflammatory mediators and suppressed macrophage infiltration., [Conclusions] Our data indicate that single local administration of dexamethasone formulated as SPT-2101 protects BLB functional integrity during endotoxemia, providing a novel therapeutic opportunity to treat diseases related to BLB dysfunction., This work was funded by a CIBERER-ISCiii SPIRALTH-CIBERER ER17PE12 and MICINN/AEI PID2020-115274RB-I00 grants to IVN and SMC and with the support of Comunidad de Madrid MINA-CM P2022-BMD-7236 and COST Action CA20121 BedBenPhar. Open Access funding provided by Consejo Superior de Investigaciones Científicas. SMC, EL, JMB and LRR held CIBERER contracts.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature., Peer reviewed

[Description of methods used for collection/generation of data] HEI-193 vestibular schwannoma cell lines, healthy human Great Auricular Nerve and vestibular schwannoma tumor samples, drug treatments, cell viability assay, In vitro senescence associated B-galactosidase activity assay, cell cycle analysis, western blotting, RNA extraction and expression analysis, annexin V-FITC and propidium iodide dual staining, immunofluorescence assays., [Background] Vestibular schwannomas (VS) are complex and heterogeneous human tumors arising from the Schwann cell compartment of the vestibulocochlear nerve. VS cause significant neurological deficit such as hearing loss and vestibular impairment, and in some cases death due to brainstem compression. There is an urgent need to find pharmacotherapies for VS since surgical removal and stereotactic radiosurgery are the only effective treatments. Cancer therapy based in the combination of drug-induced senescence and senolytics may provide an innovative pharmacological alternative for VS management., [Methods] Senescence-associated β-galactosidase (SA-β-GAL) activity detection assay, real-time polymerase chain reaction (RT-PCR), western blotting and immunofluorescence, together with viability assays were used to analyze the response to different chemotherapy drugs of the human VS HEI-193 cell line. Human VS tumor paraffin sections were also studied for SA-β-GAL-stained cells., [Results] We found that chemotherapy compounds induced genotoxic stress and cellular senescence in HEI-193 VS cells, as characterized by increased SA-β-GAL activity, growth arrest, increased levels of the cyclin-dependent kinase inhibitor p21 and the accumulation of DNA damage. These cellular senescence markers were also accompanied by an increase of senescence-associated secretory phenotype (SASP): IL6, IL8, IL1B and MMP1. Induction of senescence by chemotherapy rendered HEI-193 VS cells as druggable targets for senolytic compounds, as navitoclax. Thus, treatment with navitoclax selectively eliminated bleomycin-induced senescent HEI193 VS cells by activating the extrinsic and intrinsic apoptosis pathways. Our data also show the presence of senescent cells, SA-β-GAL-positive stain, in human VS tumors, which are not present in healthy great auricular nerve sections., [Conclusions] These findings suggest that a one-two punch strategy of pro-senescence therapy induced by chemotherapy treatment followed by senolytic therapy represents a new paradigm for the pharmacological treatment of VS., This research was funded by Spanish MCIN/ AEI/ 10.13039/ 501100011033THEARPY-PID2020-115274RB-I00,MINA-CM Madrid Innovative Neurotech Alliance P2022/BMD-7236, CA20113 COST Action EU-ProteoCure, CA23119 COST Action EU-SENESCENCE2030: Targeting Cell Senescence to Prevent Age-Related Diseases and the Spanish Senescence Network, Senestherapy (RED2018-102698-T) FEDER/MICIN grants to Isabel Varela Nieto., Figure_1_ Figure_2, Figure_3, Figure_4, Figure_5, Figure_6., Peer reviewed

[Description of methods used for collection/generation of data] ABR DATA: ABR recordings were performed on a TDT RZ6 evoked potential workstation (Tucker‐Davis Technologies, Alachua, FL, USA), as reported by Cediel et al. (2006). ABR test were performed before and after exposure to noise in G6PD transgenic and wild type mice. In brief, mice were anesthetized with ketamine (100 mg/kg; Imalgene 1000; Merial, Lyon, France) and xylazine (10 mg/kg; Rompun 2%; Bayer, Leverkusen, Germany) by intraperitoneal injection and the ABR tests were performed in a sound‐attenuating chamber. Two different sound stimuli, clicks and tone bursts, were generated with SigGenRP software (Tucker‐Davis Technologies). Stimuli were calibrated using SigCal software and an ACO Pacific 1⁄4‐inch microphone. Click stimuli were 0.1 ms and toneburst (4, 8, 16, 28, and 40 kHz) stimuli were 5‐ms duration (2.5 ms each for rise and decay, without plateau). The response was analyzed with BioSigRP software (Tucker‐Davis Technologies). Stainless steel needle electrodes were placed at the vertex and ventrolateral to the left and right ears for recording and a tweeter in open field configuration to deliver acoustic stimuli. Hearing thresholds were established at the lowest SPL level that produced a noticeable ABR five peaks wave and evoked a peak‐to‐peak voltage 2 SD above the mean background activity. Wave amplitudes, latencies, and inter‐wave latencies were determined at 70 dB SPL click stimulation.

qPCR DATA: obtained by Real-Time PCR and analyzed by QuantStudio™ Real-Time PCR software 1.3.
Cochleae were isolated from vestibules and frozen in RNAlater ® solution. Cochlear RNA extraction from pooled cochlea (n = 3 mice per experimental group), quality determination, and cDNA generation were performed as reported by Celaya et al. (2019). Quantitative amplification was performed in triplicate on an Applied Biosystems 7900HT Real‐Time PCR System using either commercial TaqMan probes or gene‐specific primers (Tables ​(Tables11 and ​and2).2). Data were collected after each amplification step and analyzed with SDS 2.2.2 software (Applied Biosystems, Foster City, CA, USA). The 18s gene was used as a housekeeping gene and the n‐fold differences were calculated using the 2–ΔΔCt method. Total G6PD expression levels are represented as the sum of the 2–ΔCt for the murine and human primers.

WB data: Whole cochleae protein extracts were prepared from 3 mice as described (Sanchez‐Calderon et al., 2010). An equal volume of extracts was resolved using SDS‐PAGE, followed by transfer to PVDF membranes (0.2 μm; Bio‐Rad Laboratories, Hercules, CA, USA) using the Bio‐Rad Trans Blot TURBO apparatus. Membranes were blocked with 5% BSA or non‐fat dried milk in 0.075% Tween, 1 mM TBS, and incubated overnight with the following antibodies:
HO-1 1:1000 Conejo Pc Millipore/ #AB1284
SOD2 1:1000 Conejo Pc Millipore/ #06-984
NQO1 1:1000 Cabra Pc Abcam/ #ab2346
NRF2 1:1000 Conejo Pc No comercial
p38 1:2000 Conejo Pc Santa Cruz/ #sc-535
p-p38 1:1000 Conejo Pc Cell Signaling/ #9211
Vinculina 1:15000 Ratón Mc Santa Cruz/ #sc-73614
IgG de cabra 1:5000 Conejo Bio-Rad Laboratories/ #1721034
IgG de conejo 1:3000 Cabra Bio-Rad Laboratories/ #1706515
IgG de ratón 1:3000 Cabra Bio-Rad Laboratories/ #1706516
Membranes were then incubated with a peroxidase‐conjugated secondary antibody for 1 h at room temperature, and bands were visualized using Clarity™ Western ECL Substrate (Bio‐Rad) using an ImageQuant LAS4000 mini digital camera (GE Healthcare Bio‐Sciences, Pittsburgh, PA, USA) and densities were quantified using Image Quant TL software.

ENZYMATIC ACTIVITY DATA:
The measurement of the enzyme activity of G6PD, 6PGD and SOD was performed in
mitochondrial or cytosolic extracts. Samples were manually homogenized in a buffer
extraction. The lysates were centrifuged to obtain the nuclear fraction
(precipitated). The supernatants were centrifuged to obtain the cytosolic (supernatant) fraction. The mitochondrial fraction was obtained by homogenizing the precipitated.
The homogenized precipitate is
Incubated in the extraction buffer for 30 minutes on ice and centrifuged at 13,000 rpm
for 10 minutes at 4ºC. Concentration protein of each of the cell fractions was determined using the DCTM Protein kit Assay Kit II (Bio-Rad Laboratories).
The activity of the enzymes G6PD and 6PGD was determined in the cytosolic fractions,
as reported (White et al., 2017).
The enzymatic activity of SOD was determined in the cytosolic and
using the EpiQuikTM Superoxide Dismutase Activity/Inhibition Assay
Kit (EpiGentek).
Optical density was measured with the VERSAmaxTM spectrophotometer
Tunable Microplate Reader using SOFTmax® Pro 3.0 Software, THEARPY: bases genéticas y moleculares de la sordera neurosensorial y del daño auditivo: exploración de nuevas dianas y estrategias terapéuticas”. Convocatoria 2020 Proyectos de I+D+i - RTI Tipo B (PID2020-115274RB-I00). FEDER/MICIN 266.200€, 2021-2024. IPs: Isabel Varela Nieto (IVN) & Silvia Murillo Cuesta, CSIC., 1) ABR DATA RAW DATA (.arf files obtained along the experiment with the Tucker Davis Tecnologies (TDT) Auditory potential workstation). RESULTS (txt, excel and SPSS files with the data obtained after ABR register analysis). 2) qPCR DATA RAW DATA (excel and jpg files obtained from the qPCR equipment) RESULTS (Analysis SYBR Cloud, Analysis TQ Cloud, Dissociation curves) 3) WB DATA RAW DATA (TIFF files of HO1, NQO1, NRF2, P22PHOX, p38, p-p38 p-JNK and SOD2 Western blotting detection). WB REPORTS (PDF files with the reports obtained with ImageQuantTM TL (GE Healthcare) software. RESULTS (Excel file with WB quantification, SPSS files with statistic analysis). 4) ENZYMATIC ACTIVITY DATA RAW DATA (Excel file with results of G6PD, 6PGD and SOD enzymatic activity). ANALYSIS (SPSS and PDF files with statistic analysis), Peer reviewed

Developmental senescence is a form of programmed senescence that contributes to morphogenesis during embryonic development. We showed recently that the SIX1 homeoprotein, an essential regulator of organogenesis, is also a repressor of adult cellular senescence. Alterations in the SIX/EYA pathway are linked to the human branchio-oto-renal (BOR) syndrome, a rare congenital disorder associated with defects in the ears, kidneys and branchial arches. Here, we have used Six1-deficient mice, an animal model of the BOR syndrome, to investigate whether dysfunction of senescence underpins the developmental defects associated with SIX1 deficiency. We have focused on the developing inner ear, an organ with physiological developmental senescence that is severely affected in Six1-deficient mice and BOR patients. We show aberrant levels and distribution of senescence markers in Six1-deficient inner ears concomitant with defective morphogenesis of senescent structures. Transcriptomic analysis and ex vivo assays support a link between aberrant senescence and altered morphogenesis in this model, associated with deregulation of the TGFβ/BMP pathway. Our results show that misregulation of embryo senescence may lead to genetic developmental disorders, significantly expanding the connection between senescence and disease., This work was supported by a grant from the Comunidad de Madrid (P2022/BMD-7393 to I.P.), and grants from Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación and the European Regional Development Fund ‘A way of making Europe’ (RTI2018-098520-B-I00 and PID2021-122600OB-I00 to I.P.; PID2020-115274RB-I00 to I.V.-N.). Open Access funding provided by Consejo Superior de Investigaciones Científicas. Deposited in PMC for immediate release.

Inherited hearing impairment is a remarkably heterogeneous monogenic condition, involving hundreds of genes, most of them with very small (< 1%) epidemiological contributions. The exception is GJB2, the gene encoding connexin-26 and underlying DFNB1, which is the most frequent type of autosomal recessive non-syndromic hearing impairment (ARNSHI) in most populations (up to 40% of ARNSHI cases). DFNB1 is caused by different types of pathogenic variants in GJB2, but also by large deletions that keep the gene intact but remove an upstream regulatory element that is essential for its expression. Such large deletions, found in most populations, behave as complete loss-of-function variants, usually associated with a profound hearing impairment. By using CRISPR-Cas9 genetic edition, we have generated a murine model (Dfnb1em274) that reproduces the most frequent of those deletions, del(GJB6-D13S1830). Dfnb1em274 homozygous mice are viable, bypassing the embryonic lethality of the Gjb2 knockout, and present a phenotype of profound hearing loss (> 90 dB SPL) that correlates with specific structural abnormalities in the cochlea. We show that Gjb2 expression is nearly abolished and its protein product, Cx26, is nearly absent all throughout the cochlea, unlike previous conditional knockouts in which Gjb2 ablation was not obtained in all cell types. The Dfnb1em274 model recapitulates the clinical presentation of patients harbouring the del(GJB6-D13S1830) variant and thus it is a valuable tool to study the pathological mechanisms of DFNB1 and to assay therapies for this most frequent type of human ARNSHI., This work was supported by the Instituto de Salud Carlos III (ISCIII), Madrid, Spain, National Plan for Scientific and Technical Research and Innovation 2017–2020, with cofunding from the European Regional Development Fund, “A way to make Europe”, grant number PI20/00619 (to I. d. C.); by MINECO/FEDER THERAPY PID2020-115274RB-I00/AEI/10.13039/501100011033, CM IND2020/BMD-17454 and MINA-CM P2022/BMD7236 (to IVN); and by MINECO BIO2015-70978-R and intramural funds from Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) (to L.M.). SMC and BMB hold CIBERER-ISCIII contracts., Peer reviewed

Hearing loss is a common side effect of many tumor treatments. However, hearing loss can also occur as a direct result of certain tumors of the nervous system, the most common of which are the vestibular schwannomas (VS). These tumors arise from Schwann cells of the vestibulocochlear nerve and their main cause is the loss of function of NF2, with 95 % of cases being sporadic and 5 % being part of the rare neurofibromatosis type 2 (NF2)-related Schwannomatosis. Genetic variations in NF2 do not fully explain the clinical heterogeneity of VS, and interactions between Schwann cells and their microenvironment appear to be critical for tumor development. Preclinical in vitro and in vivo models of VS are needed to develop prognostic biomarkers and targeted therapies. In addition to VS, other tumors can affect hearing. Meningiomas and other masses in the cerebellopontine angle can compress the vestibulocochlear nerve due to their anatomic proximity. Gliomas can disrupt several neurological functions, including hearing; in fact, glioblastoma multiforme, the most aggressive subtype, may exhibit early symptoms of auditory alterations. Besides, treatments for high-grade tumors, including chemotherapy or radiotherapy, as well as incomplete resections, can induce long-term auditory dysfunction. Because hearing loss can have an irreversible and dramatic impact on quality of life, it should be considered in the clinical management plan of patients with tumors, and monitored throughout the course of the disease., This work was funded by MICIN/FEDER THEARPY PID2020–115274RB-I00/AEI/10.13039/501100011033 (to IVN and SMC), CM IND2020/BMD-17454 (to IVN); and supported by MINA-CM P2022/BMD7236 and CONEXION NANOMEDICINA CSIC. SMC holds CIBERER-ISCIII contract., Peer reviewed

[Description of methods used for collection/generation of data] Sequencing was performed using PE150 sequencing (150 bp paired-end sequencing on each strand) on the Illumina NovaSeq X Plus platform (Illumina, San Diego, CA, USA) from Novogene. Files are row data., Los datos depositados contienen los resultados de la secuenciación masiva del ARN total aislado a partir de 63 schwannomas vestibulares, 4 schwannomas de nervios distintos al nervio vestibular (VS11, VS33, VS34 y VS74) y 7 fragmentos de diferentes nervios sanos (VS56, VS58, VS65, VS66, VS67, VS68, VS99). Los fragmentos de VS conservados en RNAlater® a -80°C se trataron con el reactivo de lisis Qiazol para la extracción de ARN utilizando el mini kit RNeasy (Qiagen, 50974106) siguiendo las instrucciones recomendadas por el fabricante en el servicio de Génomica del Instituto de Investigaciones Biomédicas Sols-Morreale de Madrid. Se utilizó ARN con un RIN (índice de integridad del ARN) superior a 7 para la secuenciación. La secuenciación se llevó a cabo utilizando la secuenciación PE150 (secuenciación “paired-end” de 150 pb en cada hebra) en la plataforma Illumina NovaSeq X Plus (Illumina, San Diego, CA, EE.UU.) de la empresa Novogene. Los datos de secuenciación depositados se presentan en formato fastq., This research was funded by Spanish MCIN PID2020-115274RB-I00 (THEARPY), PID2023-147347OB-I00 (PROHEAR), and CSIC #2024AEP115 grants to Isabel Varela-Nieto and Silvia Murillo-Cuesta, ## FICHERO X204SC24075390-Z01-F003_1.tar ##MD5-manual.pdfMD5.txtcheckSize.xlsMD5-win.exe03.Result_X204SC24075390-Z01-F002_Homo_sapiens.zipReadme.html## FICHERO X204SC24075390-Z01-F003_2.tar 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FICHERO X204SC24075390-Z01-F003_3.tar 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FICHERO X204SC24075390-Z01-F003_4.tar ##04.Ref/genome.gtf.gz04.Ref/genome_gene.fa.gz04.Ref/genome.fa.gz, Peer reviewed

Vestibular Schwannoma fragments kept in RNAlater at -80°C were treated with Qiazol lysis reagent for RNA extraction using RNeasy mini kit (Qiagen, 50974106) following the instructions recommended by the manufacturer. Human Schwann Cell RNA (#1705, ScienCell, Carlsbad, CA, USA) was used as control condition., [EN]: Fragments of human vestibular schwannomas (VS) were obtained after surgery at the Hospital Universitario de la Paz, Madrid, Spain. They were stored under specific conditions to obtain RNA. A commercial Schwann cell (SC) RNA was analysed together with these samples. The bioinformatic analysis began by mapping the sequencing reads to a reference transcriptome, then quantifying the expression levels of individual genes, and finally identifying specific genes that were differentially expressed between VS samples and SC., [ES]: Se obtuvieron fragmentos de schwannomas vestibulares (EV) humanos tras una intervención quirúrgica en el Hospital Universitario de la Paz, Madrid, España. Se almacenaron en condiciones específicas para obtener ARN. Junto con estas muestras se analizó un ARN comercial de células de Schwann (SC). El análisis bioinformático comenzó con el mapeo de las lecturas de secuenciación a un transcriptoma de referencia, luego se cuantificaron los niveles de expresión de genes individuales y, finalmente, se identificaron genes específicos que se expresaban diferencialmente entre las muestras de VS y SC., Data collection was funded by Spanish MCIN/ AEI/ 10.13039/ 501100011033 THEARPY-PID2020-115274RB-I00, MINA-CM Madrid Innovative Neurotech Alliance P2022/BMD-7236, CA20113 COST Action EU- ProteoCure, CA23119 COST Action EU-SENESCENCE2030: Targeting Cell Senescence to Prevent Age-Related Diseases and the Spanish Senescence Network grants to Isabel Varela Nieto., Genes_expresados_05_Tumor_SC.xlsl, No

[Background] Familial Meniere’s disease (FMD) is a rare polygenic disorder of the inner ear. Mutations in the connexin gene family, which encodes gap junction proteins, can also cause hearing loss, but their role in FMD is largely unknown., [Methods] We retrieved exome sequencing data from 94 individuals in 70 Meniere's disease (MD) families. Through gene burden analysis, we calculated the enrichment of rare variants (allele frequency < 0.05) in connexins genes in FMD individuals compared with the reference population. The connexin monomer and the homomeric connexon structural models were predicted using AlphaFold2 and HDOCK. RT-qPCR and immunofluorescence were done in mice cochleae to identify expression of the mouse ortholog candidate gene Gjd3., [Results] We found an enrichment of rare missense variants in the GJD3 gene when comparing allelic frequencies in FMD (N = 94) with the Spanish reference population (OR = 3.9[1.92–7.91], FDR = 2.36E-03). In the GJD3 sequence, we identified a rare haplotype (TGAGT) composed of two missense, two synonymous, and one downstream variant. This haplotype was found in five individuals with FMD, segregating in three unrelated families with a total of ten individuals; and in another eight MD individuals. GJD3 encodes the gap junction protein delta 3, also known as human connexin 31.9 (Cx31.9). The protein model predicted that the NP_689343.3:p.(His175Tyr) missense variant could modify the interaction between connexins and the connexon assembly, affecting the homotypic GJD3 gap junction between cells. Our studies in mice revealed that Gjd3—encoding Gjd3 or mouse connexin 30.2 (Cx30.2)—was expressed in the organ of Corti and vestibular organs, particularly in the tectorial membrane, the base of inner and outer hair cells and the nerve fibers., [Conclusions] The present results describe a novel association between GJD3 and FMD, providing evidence that FMD is related to changes in the inner ear channels, and supporting a new role of tectorial membrane proteins in MD., JALE has received funds to support research on genetics in Meniere’s disease from The University of Sydney (K7013_B3413 Grant), Asociacion Sindrome de Meniere España (ASMES), and Meniere’s Society, UK. AGM has received funds from the Andalusian Health Department (Grant PI-0266–2021) and from CuresWithinReach and the Knight Family. JALE and AGM have received funds from the Andalusian Government (CECEU 2020, grant code: DOC_01677). IVN and SMC have received funds from PID2020-115274RB-I00 and PID2023-147347OB-I00 funded by MCIN/AEI/https://doi.org/10.13039/501100011033, MICIU/AEI/https://doi.org/10.13039/501100011033 and FEDER, UE; and COST Action CA20113 PROTEOCURE. AMPP was supported by a PhD scholarship from the Regional Ministry of Economic Transformation, Industry, Knowledge and Universities of Junta de Andalucía (Grant number PREDOC2021/00343)., Peer reviewed

[Background] Vestibular schwannomas (VS) are complex and heterogeneous human tumors arising from the Schwann cell compartment of the vestibulocochlear nerve. VS cause significant neurological deficit such as hearing loss and vestibular impairment, and in some cases death due to brainstem compression. There is an urgent need to find pharmacotherapies for VS since surgical removal and stereotactic radiosurgery are the only effective treatments. Cancer therapy based in the combination of drug-induced senescence and senolytics may provide an innovative pharmacological alternative for VS management., [Methods] Senescence-associated β-galactosidase (SA-β-GAL) activity detection assay, real-time polymerase chain reaction (RT-PCR), western blotting and immunofluorescence, together with viability assays were used to analyze the response to different chemotherapy drugs of the human VS HEI-193 cell line. Human VS tumor paraffin sections were also studied for SA-β-GAL-stained cells., [Results] We found that chemotherapy compounds induced genotoxic stress and cellular senescence in HEI-193 VS cells, as characterized by increased SA-β-GAL activity, growth arrest, increased levels of the cyclin-dependent kinase inhibitor p21 and the accumulation of DNA damage. These cellular senescence markers were also accompanied by an increase of senescence-associated secretory phenotype (SASP): IL6, IL8, IL1B and MMP1. Induction of senescence by chemotherapy rendered HEI-193 VS cells as druggable targets for senolytic compounds, as navitoclax. Thus, treatment with navitoclax selectively eliminated bleomycin-induced senescent HEI-193 VS cells by activating the extrinsic and intrinsic apoptosis pathways. Our data also show the presence of senescent cells, SA-β-GAL-positive stain, in human VS tumors, which are not present in healthy great auricular nerve sections., [Conclusions] These findings suggest that a one-two punch strategy of pro-senescence therapy induced by chemotherapy treatment followed by senolytic therapy represents a new paradigm for the pharmacological treatment of VS., This research was funded by Spanish MCIN/ AEI/ 10.13039/ 501100011033 THEARPY-PID2020-115274RB-I00, MINA-CM Madrid Innovative Neurotech Alliance P2022/BMD-7236, CA20113 COST Action EU- ProteoCure, CA23119 COST Action EU-SENESCENCE2030: Targeting Cell Senescence to Prevent Age-Related Diseases and the Spanish Senescence Network, Senestherapy (RED2018-102698-T) FEDER/ MICIN grants to I. V. -N., Peer reviewed

[Objetivo]: Los schwannomas vestibulares (SV) son tumores benignos de comportamiento clínico variable. La recidiva tras la cirugía es infrecuente, aunque en ocasiones agresiva. No se dispone de predictores moleculares fiables de recidiva, y la correlación genotipo-fenotipo es débil. Este estudio tuvo como objetivo identificar marcadores moleculares asociados con la recidiva en SV y explorar su potencial pronóstico., [Métodos]: Estudio retrospectivo de casos y controles en pacientes intervenidos quirúrgicamente de SV entre 2017 y 2024. Se realizó RNA-Seq para identificar firmas moleculares asociadas con la recidiva, y los genes seleccionados se validaron mediante RT-qPCR. Se analizó la supervivencia libre de recidiva y la supervivencia libre de cirugía de rescate en función de la extensión de la resección., [Resultados]: Se analizaron 23 muestras procedentes de 22 pacientes, incluyendo 7 casos de recurrencia y 16 de no recurrencia. Se identificaron 81 genes diferencialmente expresados en los tumores que presentaron recurrencia, con enriquecimiento de las vías MAPK (FDR = 7,9 × 10⁻⁴) y PI3K/AKT/mTOR (FDR = 0,038). Se seleccionaron ocho genes candidatos para su validación: DCN (FC 4,34); DUSP5 (FC 4,20); CXCL8 (FC 3,25); JUND (FC 3,27); MYC (FC 2,45); MAFF (FC 3,79); SOD3 (FC 2,47) (todos sobreexpresados), y TNF (FC 0,16) (infraexpresado). La RT-qPCR confirmó que CXCL8 y TNF se encontraban significativamente elevados en los tumores que requirieron cirugía de rescate precoz, así como en un caso de Schwannomatosis asociada a NF2. No se observaron diferencias en la supervivencia libre de recidiva ni en la supervivencia libre de cirugía de rescate entre las resecciones casi totales y las subtotales., [Conclusión]: Los SV que presentan recurrencia muestran un perfil molecular diferenciado, en el que destacan IL-8 y TNF como potenciales biomarcadores y dianas terapéuticas. Estos hallazgos apoyan el desarrollo de un panel pronóstico que permita guiar un seguimiento y tratamiento personalizados, en particular en pacientes de alto riesgo. Son necesarios estudios adicionales antes de su implementación clínica., This research was funded by Spanish MCIN/AEI/10.13039/501100011033 THEARPY-PID2020-115274RB-I00 and MICIU/AEI/10.13039/501100011033 PROHEAR-PID2023-147347OB-I00 to IVN. The authors thank the support from Madrid Innovative Neurotech Alliance MINA-CM P2022/BMD-7236 consortium and CSIC Rare Diseases Network (RER-Biomed)., No

[Purpose] Vestibular schwannomas (VS) are benign tumors with variable clinical behavior. Recurrence after surgery is uncommon yet sometimes aggressive. Reliable molecular predictors of recurrence are lacking, and the correlation between genotype and phenotype is weak. This study aimed to identify molecular markers associated with recurrence in VS and explore their prognostic potential., [Methods] Retrospective case-control study of patients undergoing VS surgery between 2017 and 2024. RNA-Seq was conducted to identify molecular signatures associated with recurrence, and selected genes were validated using RT-qPCR. Recurrence-free and salvage surgery-free survival were analyzed by the extent of resection., [Results] 23 samples from 22 patients were analyzed, including 7 recurrent and 16 non-recurrent cases. 81 differentially expressed genes were identified in recurrent tumors, with enrichment of the MAPK (FDR = 7.9 × 10⁻⁴) and PI3K/AKT/mTOR (FDR = 0.038) pathways. Eight candidate genes were selected for validation: DCN (FC 4.34); DUSP5 (FC 4.20); CXCL8 (FC 3.25); JUND (FC 3.27); MYC (FC 2.45); MAFF (FC 3.79); SOD3 (FC 2.47) (all upregulated), and TNF (FC 0.16) (downregulated). RT-qPCR confirmed that CXCL8 and TNF were significantly elevated in tumors requiring early salvage surgery and in an NF2-associated Schwannomatosis case. No differences in recurrence-free or salvage surgery-free survival were observed between near-total and subtotal resections., [Conclusion] Recurrent VS exhibit a distinct molecular profile, highlighting IL-8 and TNFα as potential biomarkers and therapeutic targets. These findings support the development of a prognostic panel to guide personalized follow-up and treatment, particularly for high-risk patients. Further studies are needed before clinical implementation., This research was funded by Spanish Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación MCIN/AEI/https://doi.org/10.13039/501100011033 THEARPY-PID2020-115274RB-I00 and Spanish Ministerio de Ciencia, Innovación y Universidades/Agencia Estatal de Investigación MICIU/AEI/https://doi.org/10.13039/501100011033 PROHEAR-PID2023-147347OB-I00 to Isabel Varela-Nieto. The authors thank the support from Madrid Innovative Neurotech Alliance MINA-CM P2022/BMD-7236 consortium and CSIC Rare Diseases Network (RER-Biomed)., Peer reviewed

Primary Schwann cell cultures derived from rat vestibulocochlear and sciatic nerves, drug treatments, cell viability assay, senescence associated B-galactosidase activity assay, RNA extraction and expression analysis, immunofluorescence assays., Schwann cells are the principal glial cells of the peripheral nervous system and play essential roles in myelination and axonal conduction. These cells can acquire a senescent cellular programme, which can contribute to their functional decline. Schwann cells have been also implicated in vestibular schwannoma, a common cause of sensorineural hearing loss. Notably, it must be highlighted that cellular senescence might serve as an important tumour-suppressive mechanism. In this study, we isolated and characterized primary Schwann cells from the rat vestibulocochlear nerve (VCN), revealing distinct morphological and biomolecular features. During prolonged in vitro expansion, VCN-derived Schwann cells maintained stable morphology up to late passages but showed progressive downregulation of myelin-associated genes and transcription factors, alongside increased expression of cell-cycle inhibitors (e.g. Cdkn2a, p53) and senescence-associated-related genes (e.g. Ccl2, Il1b), indicating the acquisition of a senescence-associated phenotype. Treatment with etoposide confirmed that these cells retain a fully functional senescence program, as shown by reduced proliferation, increased SA-β-Gal positivity, and upregulation of senescence markers. Interestingly, the Schwann cell mitogen neuregulin-1, promoted their senescence-associated phenotype, driving cytoskeletal remodelling, altering the expression of Schwann cell specific genes, and enhancing senescence markers at later passages. Overall, this study establishes VCN-derived Schwann cells as a reliable model for studying the senescence mechanisms and highlights the capacity of neuregulin-1 as a potential pro-senescence modulator of Schwann cells., This research was funded by Spanish MCIN/ AEI/ 10.13039/ 501100011033 THEARPY-PID2020-115274RB-I00 and CA23119 COST Action EU-SENESCENCE2030: Targeting Cell Senescence to Prevent Age-Related Diseases to Isabel Varela Nieto and Silvia Murillo Cuesta. It was also supported by Progetto di Eccellenza 2023–2027, from the Italian Ministry of University and Research (MUR), and Children Tumor Foundation (CTF) US to Valerio Magnaghi., Peer reviewed

MCIN PID2020-115274RB-I00 and PID2023-147347OB-I00 (to S.M.-C. and I.V.-N.)
MCIN PID2021-127860OB-I00 and PID2024-156098OB-I00 (to J.G.P.)
Fundación Rioja Salud (Gobierno de La Rioja, Spain), co-funded by the ERDF/ESF, FEDER011_2024/ID-1813 (to J.G.P.)
MCIN PRE2021-101114 (predoctoral contract to C.G.-M.)
Fundación Científica AECC PRDLR258489BENI (predoctoral fellowship to J.B.-L.)
European Research Council (ERC) Starting Grant RADIOGUT (Grant Agreement No. 101040951) and SFI/IRC Pathway Award 21/PATH-S/9424 (to M.R.A.), Peer reviewed