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

[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

[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

[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

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