BUSCADOR RECOLECTA

© 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.

Table of contents: 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], MCIN/AEI/10.13039/ 501100011033 THEARPY-PID2020-115274RB-I00; 0551_PSL_6_E POCTEP FGCSIC/ PSL-INTERREG/FEDER NITROPROHEAR, 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

[Description of methods used for collection/generation of data] ABR DATA: obtained by ABR recording in a Tucker David Technology RZ6 Auditory Workstation using BioSigRZ software qPCR DATA: obtained by Real-Time PCR and analyzed by QuantStudio™ Real-Time PCR software 1.3., Oxidative stress is a major triggering factor for noise-induced hearing loss. Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the rate-limiting step in the pentose-phosphate pathway, and is a key molecule in the cellular antioxidant response. G6PD overexpression in the transgenic mouse delays the onset of age-related hearing loss. However, its antioxidant potential against noise has not been explored yet. In this work, it has been shown that G6PD overexpression transiently protects against hearing damage caused by noise exposure, through activation of the antioxidant response mediated by NRF2-NQO1 and SOD, 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, ) 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), 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] Freshly harvested samples of sporadic VS were obtained from indicated surgeries and transported to the laboratory on ice. All protocols involving patient samples were approved by the local Ethics Review Board of La Paz University Hospital and the CSIC, and were conducted in accordance with the Helsinki Declaration. Written informed consent was obtained from all participants. Freshly harvested VS tissue was rinsed several times and minced under sterile conditions into sub-fragments, which were then dissociated with and enzymatic digestion. Cells were cultured i for 2–3 weeks. In addition, we used an establish cell line called HEI-193 cell line, derived from a VS of a patient with NF2., [Methods for processing the data] To generate the data we performed different experimental techniques: cell viability assays using crystal violet staining; analysis of B-galactosidase enzyme activity associated with senescence; cell cycle analysis using PI staining in the flow cytometer; study of protein and mRNA expression by Western blotting and RT-qPCR, respectively; analysis of the numer of apoptotic cells using Annexin V-FITC and propidium iodide dual staining., Cellular senescence is a crucial response against cancer development. It has been demonstrated that this cellular state is a defining feature of premalignant tumors that could be important in cancer diagnosis. This tumor-supressor role of cellular senescence has not been studied so far in human vestibular schwannomas (VS). VS are benign tumors originating from the Schwann cells that form the myelin sheath of the cochleovestibular nerve. VS grow slowly and cause hearing loss both due to compression of the auditory nerve and the release of ototoxic substances. Currently, patients undergo periodic MRI, surgery, gamma-radiosurgery or radiotherapy as tumor treatment but there are no pharmacological FDA-approved therapies to treat these tumors. In this study, we used HEI-193 cell cultures and patient derived VS primary cell cultures to address whether conventional chemotherapy, mainly DNA damage drugs, may induce a senescent response in these cellular systems that makes cells susceptible to be targeted by senolytic agents. Our data show that bleomycin can induce several senescent markers in VS primary cell culture as well as in an established cell line derived from a patient with neurofibromatosis type II, HEI-193 cell line. We analyzed the SASP-related secretory profile and observed that VS expressed higher levels of cytokines, metalloproteases and other SASP components than those obtained in bleomycin-treated HEI-193 cells. Our results also show that treatment with navitoclax, a senolytic agent, decreases the viability of the bleomycin- induced senescent cells without affecting the viability of the proliferative ones. This two-punch strategy based in the combination of senogenic and senolytic agents could constitute a potential alternative for the 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 and the Spanish Senescence Network, Senestherapy (RED2018-102698-T) FEDER/ MICIN grants., In the Data Acumulated_03112023 folder you will find two files: a folder called "figures" and an excel file entitled "statistics and figure details". The figures folder contains the original files and the excell files of our work, separated by figure number as designed for the scientific article we have just submitted to the journal Molecular Oncology and which I will detail later. The excel file called statistics and figure details contains as many sheets as figures we have designed for this article. The details of the brightfield and fluorescence microscopy photographs and the statistics used in each panel of each figure are described. Going back to the "figures" folder, you will find 11 subfolders where each of the panels of the figure appears with raw data and excel files., 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