Isabel Varela‐Nieto

Insulin-like growth factor-1 (IGF-1) has been shown to play a key role during embryonic and postnatal development of the CNS, but its effect on a sensory organ has not been studied in vivo. Therefore, we examined cochlear growth, differentiation, and maturation in Igf-1 gene knock-out mice at postnatal days 5 (P5), P8, and P20 by using stereological methods and immunohistochemistry. Mutant mice showed reduction in size of the cochlea and cochlear ganglion. An immature tectorial membrane and a significant decrease in the number and size of auditory neurons were also evident at P20. IGF-1-deficient cochlear neurons showed increased caspase-3-mediated apoptosis, along with aberrant expression of the early neural markers nestin and Islet 1/2. Cochlear ganglion and fibers innervating the sensory cells of the organ of Corti presented decreased levels of neurofilament and myelin P(0) in P20 mouse mutants. In addition, an abnormal synaptophysin expression in the somata of cochlear ganglion neurons and sensory hair cells suggested the persistence of an immature pattern of synapses distribution in the organ of Corti of these animals. These results demonstrate that lack of IGF-1 in mice severely affects postnatal survival, differentiation, and maturation of the cochlear ganglion cells and causes abnormal innervation of the sensory cells in the organ of Corti.

We found that natural (long-chain) ceramide 1-phosphate can be dispersed into aqueous solution when dissolved in an appropriate mixture of methanol/dodecane (49:1, v/v). This solvent mixture facilitates the interaction of this phosphosphingolipid with cells. Under these conditions, incubation of EGFR T17 fibroblasts with natural ceramide 1-phosphate caused a potent stimulation of DNA synthesis. This effect was accompanied by an increase in the levels of proliferating-cell nuclear antigen. Concentrations of natural ceramide 1-phosphate that stimulated the synthesis of DNA did not inhibit adenylate cyclase activity, nor did they stimulate phospholipase D. Natural ceramide 1-phosphate did not alter the cellular phosphorylation state of tyrosine residues or of mitogen-activated protein kinase. Furthermore, natural ceramide 1-phosphate failed to induce the expression of the proto-oncogenes c-myc and c-fos. Both the stimulation of DNA synthesis and the induction of proliferating-cell nuclear antigen by natural ceramide 1-phosphate were inhibited by natural ceramides. This work suggests that the use of methanol and dodecane to deliver natural ceramide 1-phosphate to cells may be useful for elucidation of the biological function(s) and mechanism(s) of action of ceramide 1-phosphate.

Abstract It has been reported that mutations in the gene encoding human insulin‐like growth factor‐I (IGF‐I) cause syndromic hearing loss. To study the precise role of IGF‐I in auditory function and to hypothesize the possible morphological and electrophysiological changes that may occur in the human inner ear, we have analysed the auditory brainstem response in a mouse model of IGF‐I deficiency. We show here that homozygous Igf‐1 – / – mice present an all‐frequency involved bilateral sensorineural hearing loss. Igf‐1 –/– mice also present a delayed response to acoustic stimuli; this increases along the auditory pathway, indicating a contribution of the central nervous system to the hearing loss in Igf‐1 –/– mice. These results support the use of the Igf‐1 –/– mouse as a new model for the study of human syndromic deafness.

The effects of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) on the differentiation of avian cochleovestibular ganglion and their possible association with the hydrolysis of glycosyl-phosphatidylinositol (GPI) were studied. BDNF and NT-3 (2 ng/ml) promoted neurite outgrowth in explants of both cochlear and vestibular ganglia. This effect on neuritogenesis was stage-dependent, reaching a maximum at E7 for NT-3 and at E9 for BDNF. The magnitude of the response of the vestibular ganglion to BDNF was always smaller than that of the cochlear ganglion of an equivalent stage. BDNF and NT-3 stimulation of neuronal survival and neurite extension was also demonstrated in dissociated neuronal cell cultures. The effect was concentration-dependent with saturation of the response occurring at 4 ng/ml for BDNF and at 2 ng/ml for NT-3, the half-maximal effect occurring at 2 and 1 ng/ml, respectively, for the most sensitive stages of the chick cochlear ganglion. Inositol phosphoglycan (IPG) did not mimic the effects of BDNF or NT-3 on neuronal survival and neurite outgrowth, nor was it able to potentiate their responses. Antibodies raised against IPG did not block the effects of these neurotrophins. The results suggest that BDNF and NT-3 may act in cooperation to establish the innervation pattern of the inner ear. Unlike their early proliferative effects, neurotrophic effects are uncoupled from the GPI/IPG signal transduction system.

Lysophosphatidic acid (LPA) has emerged as a new regulatory molecule in the brain. Recently, some studies have shown a role for this molecule and its LPA(1) receptor in the regulation of plasticity and neurogenesis in the adult brain. However, no systematic studies have been conducted to investigate whether the LPA(1) receptor is involved in behavior. In this study, we studied the phenotype of maLPA(1)-null mice, which bear a targeted deletion at the lpa(1) locus, in a battery of tests examining neurologic performance, habituation in exploratory behavior in response to low and mild anxiety environments and spatial memory. MaLPA(1)-null mutants showed deficits in both olfaction and somesthesis, but not in retinal or auditory functions. Sensorimotor co-ordination was impaired only in the equilibrium and grasping reflexes. The mice also showed impairments in neuromuscular strength and analgesic response. No additional differences were observed in the rest of the tests used to study sensoriomotor orientation, limb reflexes and co-ordinated limb use. At behavioral level, maLPA(1)-null mice showed an impaired exploration in the open field and increased anxiety-like response when exposed to the elevated plus maze. Furthermore, the mice exhibit impaired spatial memory retention and reduced use of spatial strategies in the Morris water maze. We propose that the LPA(1) receptor may play a major role in both spatial memory and response to anxiety-like conditions.

Nutritional imbalance is emerging as a causative factor of hearing loss. Epidemiologic studies have linked hearing loss to elevated plasma total homocysteine (tHcy) and folate deficiency, and have shown that folate supplementation lowers tHcy levels potentially ameliorating age-related hearing loss. The purpose of this study was to address the impact of folate deficiency on hearing loss and to examine the underlying mechanisms. For this purpose, 2-mo-old C57BL/6J mice (Animalia Chordata Mus musculus) were randomly divided into 2 groups (n = 65 each) that were fed folate-deficient (FD) or standard diets for 8 wk. HPLC analysis demonstrated a 7-fold decline in serum folate and a 3-fold increase in tHcy levels. FD mice exhibited severe hearing loss measured by auditory brainstem recordings and TUNEL-positive-apoptotic cochlear cells. RT-quantitative PCR and Western blotting showed reduced levels of enzymes catalyzing homocysteine (Hcy) production and recycling, together with a 30% increase in protein homocysteinylation. Redox stress was demonstrated by decreased expression of catalase, glutathione peroxidase 4, and glutathione synthetase genes, increased levels of manganese superoxide dismutase, and NADPH oxidase-complex adaptor cytochrome b-245, α-polypeptide (p22phox) proteins, and elevated concentrations of glutathione species. Altogether, our findings demonstrate, for the first time, that the relationship between hyperhomocysteinemia induced by folate deficiency and premature hearing loss involves impairment of cochlear Hcy metabolism and associated oxidative stress.

WBP2 encodes the WW domain-binding protein 2 that acts as a transcriptional coactivator for estrogen receptor α (ESR1) and progesterone receptor (PGR). We reported that the loss of Wbp2 expression leads to progressive high-frequency hearing loss in mouse, as well as in two deaf children, each carrying two different variants in the WBP2 gene. The earliest abnormality we detect in Wbp2-deficient mice is a primary defect at inner hair cell afferent synapses. This study defines a new gene involved in the molecular pathway linking hearing impairment to hormonal signalling and provides new therapeutic targets.

El schwannoma vestibular (SV) es el tumor más frecuente del ángulo pontocerebeloso. La mayor accesibilidad a las pruebas radiológicas ha incrementado su diagnóstico. Teniendo en cuenta las características del tumor, la clínica y la edad del paciente se han propuesto tres estrategias terapéuticas, observación, cirugía o radioterapia. La elección de la más adecuada para cada paciente es un motivo de controversia frecuente. El presente trabajo incluye una revisión exhaustiva sobre cuestiones relativas al SV que pueden servir de guía clínica en el manejo de pacientes con estas lesiones. La presentación se ha orientado en forma de preguntas que el clínico se hace habitualmente y las respuestas están redactadas y/o revisadas por un panel de expertos nacionales e internacionales consultados por la Comisión de Otología de la SEORL-CCC. Se ha elaborado un listado con los 13 bloques temáticos más controvertidos sobre el manejo del SV en forma de 50 preguntas y se han buscado las respuestas a todas ellas mediante una revisión sistemática de la literatura (artículos publicados en PubMed y Cochrane Library entre 1992 y 2023 sobre cada bloque temático). Treinta y tres expertos, liderados por la Comisión de Otología de la SEORL-CCC, han analizado y discutido todas las respuestas. En el Anexo 1 pueden encontrarse 14 preguntas adicionales divididas en cuatro bloques temáticos. Esta guía de práctica clínica sobre el manejo del SV ofrece respuestas consensuadas a las preguntas más habituales que se plantean sobre este tumor. La ausencia de suficientes estudios prospectivos hace que los niveles de evidencia sobre el tema sean en general medios o bajos. Este hecho incrementa el interés de este tipo de guías de práctica clínica elaboradas por expertos. Vestibular schwannoma (VS) is the most common tumour of the cerebellopontine angle. The greater accessibility to radiological tests has increased its diagnosis. Taking into account the characteristics of the tumour, the symptoms and the age of the patient, three therapeutic strategies have been proposed: observation, surgery or radiotherapy. Choosing the most appropriate for each patient is a frequent source of controversy. This paper includes an exhaustive literature review of issues related to VS that can serve as a clinical guide in the management of patients with these lesions. The presentation has been oriented in the form of questions that the clinician usually asks himself and the answers have been written and/or reviewed by a panel of national and international experts consulted by the Otology Commission of the SEORL-CCC. A list has been compiled containing the 13 most controversial thematic blocks on the management of VS in the form of 50 questions, and answers to all of them have been sought through a systematic literature review (articles published on PubMed and Cochrane Library between 1992 and 2023 related to each thematic area). Thirty-three experts, led by the Otology Committee of SEORL-CCC, have analyzed and discussed all the answers. In Annex 1, 14 additional questions divided into 4 thematic areas can be found. This clinical practice guideline on the management of VS offers agreed answers to the most common questions that are asked about this tumour. The absence of sufficient prospective studies means that the levels of evidence on the subject are generally medium or low. This fact increases the interest of this type of clinical practice guidelines prepared by experts.

Vestibular schwannoma (VS) is the most common tumour of the cerebellopontine angle. The greater accessibility to radiological tests has increased its diagnosis. Taking into account the characteristics of the tumour, the symptoms and the age of the patient, three therapeutic strategies have been proposed: observation, surgery or radiotherapy. Choosing the most appropriate for each patient is a frequent source of controversy.This paper includes an exhaustive literature review of issues related to VS that can serve as a clinical guide in the management of patients with these lesions. The presentation has been oriented in the form of questions that the clinician usually asks himself and the answers have been written and/or reviewed by a panel of national and international experts consulted by the Otology Commission of the SEORL-CCC.A list has been compiled containing the 13 most controversial thematic blocks on the management of VS in the form of 50 questions, and answers to all of them have been sought through a systematic literature review (articles published on PubMed and Cochrane Library between 1992 and 2023 related to each thematic area). Thirty-three experts, led by the Otology Committee of SEORL-CCC, have analyzed and discussed all the answers. In Annex 1, 14 additional questions divided into 4 thematic areas can be found.This clinical practice guideline on the management of VS offers agreed answers to the most common questions that are asked about this tumour. The absence of sufficient prospective studies means that the levels of evidence on the subject are generally medium or low. This fact increases the interest of this type of clinical practice guidelines prepared by experts.

Hearing and balance receptors in the inner ear are highly susceptible to damage caused by a wide variety of toxic substances, including aminoglycosides. This class of antibiotics is commonly used in medicine, even though they may produce irreversible bilateral neurosensorial deafness. To identify potential ototoxic agents and novel therapeutic targets, it is necessary to generate standardized animal models of aminoglycoside ototoxicity, which will also serve to explore otic cell repair and regeneration. Although the mouse is the species most often used in biomedical research, due to the genetic information and genetically-modified strains available, there are few standard models of aminoglycoside ototoxicity in adult mice. Most protocols to produce ototoxicity in adult mice employ high doses of aminoglycosides for long periods of time, which causes systemic toxicity, side-effects and high mortality rates. Here, we compare the effects of systemic treatment with four different, yet common, aminoglycoside antibiotics in two mouse strains, evaluating their effects on mortality, cochlear morphology and auditory brainstem responses. Our data indicate that gentamicin and neomycin caused high mortality in the adult mouse without significantly changing the auditory threshold. Amikacin produced a tolerable rate of mortality but at doses that did not exhibit ototoxicity. Finally, intramuscular injection of kanamycin in C57BL/6JOlaHsd mice induced significant dose-dependent bilateral hearing loss with a moderate rate of mortality and less discomfort than following subcutaneous administration.

Hearing aids or cochlear implants constitute almost exclusively the treatment options currently available to patients suffering from sensorineural hearing loss and related conditions, such as noise-induced hearing loss, ototoxicity or autoimmune inner ear disease. While some systemic treatments exist, they generally exert adverse secondary effects and their efficacy is hampered by the blood-cochlear barrier that limits drug access to the inner ear. Hence, the new therapies that are being developed for hearing loss focus on strategies for direct drug delivery to the inner ear. The passive and active methods for local delivery can be categorized into two general groups: intratympanic or intracochlear. The intratympanic approach is a non-invasive method that preserves hearing and takes advantage of the permeability of the round window to gain access to the cochlea. However, this technique is limited by not knowing the dose of the drug that reaches the cochlea, (a handicap which might be overcome by the use of tagged drugs). While direct access to the inner ear by intracochlear administration avoids this problem, this method requires surgery. Currently, laboratory animals are being used to explore which therapeutic approaches are best suited to address this problem. These include cochleostomy and the insertion of devices that pump drugs into the inner ear without inducing cochlear damage. In this article, we review the different techniques under evaluation in animal models of deafness, and their potential use for drug delivery and treatment of human inner ear diseases.

Autophagy is an evolutionarily conserved catabolic process by which cells degrade their own components through the lysosomal machinery. In physiological conditions, the mechanism is tightly regulated and contributes to maintain a balance between synthesis and degradation in cells undergoing intense metabolic activities. Autophagy is associated with major tissue remodeling processes occurring through the embryonic, fetal and early postnatal periods of vertebrates. Here we survey current information implicating autophagy in cellular death, proliferation or differentiation in developing vertebrates. In developing systems, activation of the autophagic machinery could promote different outcomes depending on the cellular context. Autophagy is thus an extraordinary tool for the developing organs and tissues.

In a Dutch consanguineous family with recessively inherited nonsyndromic hearing impairment (HI), homozygosity mapping combined with whole-exome sequencing revealed a MPZL2 homozygous truncating variant, c.72del (p.Ile24Metfs∗22). By screening a cohort of phenotype-matched subjects and a cohort of HI subjects in whom WES had been performed previously, we identified two additional families with biallelic truncating variants of MPZL2. Affected individuals demonstrated symmetric, progressive, mild to moderate sensorineural HI. Onset of HI was in the first decade, and high-frequency hearing was more severely affected. There was no vestibular involvement. MPZL2 encodes myelin protein zero-like 2, an adhesion molecule that mediates epithelial cell-cell interactions in several (developing) tissues. Involvement of MPZL2 in hearing was confirmed by audiometric evaluation of Mpzl2-mutant mice. These displayed early-onset progressive sensorineural HI that was more pronounced in the high frequencies. Histological analysis of adult mutant mice demonstrated an altered organization of outer hair cells and supporting cells and degeneration of the organ of Corti. In addition, we observed mild degeneration of spiral ganglion neurons, and this degeneration was most pronounced at the cochlear base. Although MPZL2 is known to function in cell adhesion in several tissues, no phenotypes other than HI were found to be associated with MPZL2 defects. This indicates that MPZL2 has a unique function in the inner ear. The present study suggests that deleterious variants of Mplz2/MPZL2 affect adhesion of the inner-ear epithelium and result in loss of structural integrity of the organ of Corti and progressive degeneration of hair cells, supporting cells, and spiral ganglion neurons. In a Dutch consanguineous family with recessively inherited nonsyndromic hearing impairment (HI), homozygosity mapping combined with whole-exome sequencing revealed a MPZL2 homozygous truncating variant, c.72del (p.Ile24Metfs∗22). By screening a cohort of phenotype-matched subjects and a cohort of HI subjects in whom WES had been performed previously, we identified two additional families with biallelic truncating variants of MPZL2. Affected individuals demonstrated symmetric, progressive, mild to moderate sensorineural HI. Onset of HI was in the first decade, and high-frequency hearing was more severely affected. There was no vestibular involvement. MPZL2 encodes myelin protein zero-like 2, an adhesion molecule that mediates epithelial cell-cell interactions in several (developing) tissues. Involvement of MPZL2 in hearing was confirmed by audiometric evaluation of Mpzl2-mutant mice. These displayed early-onset progressive sensorineural HI that was more pronounced in the high frequencies. Histological analysis of adult mutant mice demonstrated an altered organization of outer hair cells and supporting cells and degeneration of the organ of Corti. In addition, we observed mild degeneration of spiral ganglion neurons, and this degeneration was most pronounced at the cochlear base. Although MPZL2 is known to function in cell adhesion in several tissues, no phenotypes other than HI were found to be associated with MPZL2 defects. This indicates that MPZL2 has a unique function in the inner ear. The present study suggests that deleterious variants of Mplz2/MPZL2 affect adhesion of the inner-ear epithelium and result in loss of structural integrity of the organ of Corti and progressive degeneration of hair cells, supporting cells, and spiral ganglion neurons.

Among metabolic diseases, diabetes is considered one of the most prevalent throughout the world. Currently, statistics show that over 10% of the world's aged population (60 years and older) suffers from diabetes. As a consequence, it consumes a considerable proportion of world health expenditure. This review considers both past and current research into the molecular basis of insulin resistance found in type II diabetes and focuses on the role of inositol-containing phospholipid metabolism. It has been firmly established that the activation of phosphatidylinositol 3-kinase (PI3-K) is important for the propagation of the metabolic actions of insulin. In addition to the 3-phosphorylated phosphatidylinositols formed via the action of PI3-K, the glycosyl-phosphatidylinositol/inositol phosphoglycan (GPI/IPG) signaling component is also strongly implicated in mediating numerous metabolic actions of insulin. Although all the elements within the type II diabetes phenotype have not been fully defined, it has been proposed that defects in insulin transmembrane signaling through malfunction of inositol-containing phospholipid metabolism and absenteeism of the generation of phospholipid-derived second messengers may be associated with the appearance of the type II diabetic phenotype. Pharmaceutical approaches using synthetically produced IPG analogues, which themselves mimic insulin's actions, alone or in combination with other drugs, may lead the way toward introducing alternative therapies for type II diabetes in the coming years.

Abstract This is a review of the biological processes and the main signaling pathways required to generate the different otic cell types, with particular emphasis on the actions of insulin‐like growth factor I. The sensory organs responsible of hearing and balance have a common embryonic origin in the otic placode. Lineages of neural, sensory, and support cells are generated from common otic neuroepithelial progenitors. The sequential generation of the cell types that will form the adult inner ear requires the coordination of cell proliferation with cell differentiation programs, the strict regulation of cell survival, and the metabolic homeostasis of otic precursors. A network of intracellular signals operates to coordinate the transcriptional response to the extracellular input. Understanding the molecular clues that direct otic development is fundamental for the design of novel treatments for the protection and repair of hearing loss and balance disorders. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.

Nerve growth factor (NGF) exerts a variety of actions during embryonic development. At the early stages of inner ear development, NGF stimulates cell proliferation, an effect mediated through low-affinity receptors. We have studied the possibility that the glycosyl-phosphatidylinositol/inositol phosphoglycan (glycosyl-PtdIns/IPG) system is involved in transmitting this NGF signal. Endogenous glycosyl-PtdIns was characterized in extracts of cochleovestibular ganglia (CVGs) that incorporated [3H]glucosamine, [3H]galactose, [3H]myristic acid, and [3H]palmitic acid. Incubation of CVG with NGF produced a rapid and transient hydrolysis of glycosyl-PtdIns. Hydrolysis was complete at 100 ng/ml, and the half-maximal effect occurred at 25 ng/ml, overlapping with the concentration dependence of the mitogenic effect of NGF. An IPG was isolated from embryonic extracts. It had biological effects similar to those reported for the insulin-induced IPG in other tissues. It exerted a powerful mitogenic effect on CVG, comparable to that of NGF. Both the IPG- and NGF-induced cell proliferation were blocked by anti-IPG antibodies that recognized the endogenous IPG on a silica plate immunoassay. These results show that CVG possesses a fully active glycosyl-PtdIns/IPG signal transduction system and that the proliferative effects associated with NGF binding to low-affinity receptors require IPG generation.

Sensorineural hearing loss is a clinical heterogeneous disorder and a significant health-care problem with tremendous socio-economic impact. According to WHO, "Over 5% of the world's population has disabling hearing loss -328 million adults and 32 million children-". In children, early hearing loss affects language acquisition. Hearing deficits are generally associated with the loss of the sensory "hair" cells and/or neurons caused by primary genetic defects or secondary to environmental factors including infections, noise and ototoxic drugs. Hearing loss cannot be reversed and currently the available treatment is limited to hearing aids and cochlear implants. Studies are being conducted to develop alternative treatments combining both preventive and reparative strategies. Human insulin like growth factor (IGF) I deficiency is a rare disease associated with hearing loss, poor growth rates and mental retardation (ORPHA73272, OMIM608747). Similarly, lgf1-/- mice are dwarfs with poor survival rates and congenital profound sensorineural deafness. IGF-I is known to be a neuroprotective agent that maintains cellular metabolism, activates growth, proliferation and differentiation, and limits cell death. Here we will discuss the basic mechanisms underlying IGF-I actions in the auditory system and their clinical implications to pursue novel treatments to ameliorate hearing loss.

The signaling cascade Ras/Raf/mitogen-activated protein kinases modulates cell proliferation, differentiation, and survival, all key cellular processes during neural development. To better define the in vivo role of Raf during chick retinal neurogenesis, we interfered with Raf-dependent signaling during days 4.5 to 7.5 of embryonic development by expressing a dominant negative mutant of c-Raf (DeltaRaf), which blocks Ras-dependent Raf activation, and by overexpressing wild-type c-Raf. DeltaRaf expression induced an increase in cell death by apoptosis, whereas it did not affect overall cell proliferation and differentiation. In parallel, the number of Islet-1/2-positive and TUJ1-positive retinal ganglion cells were diminished in their definitive layer, whereas there was an increase in the number of mislocated Islet-1/2-positive cells. This disturbed morphogenesis correlated with a disruption of the optic fiber layer. Conversely, c-Raf overexpression caused moderate opposite effects on apoptosis. These results frame in vivo early neurogenesis processes in which c-Raf is essential.

We have investigated the role of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) in the regulation of cell proliferation in the early developing cochleovestibular ganglion (CVG). Ganglia were isolated from 72-hr chick embryos and cultured for 24 hr. Both BDNF and NT-3 had a powerful mitogenic effect, at doses of 1-5 ng/ml, consistent with an involvement of the high-affinity receptor. Evidence for the participation of the glycosyl-phosphatidylinositol (GPI)/inositol phosphoglycan (IPG) signaling system in the mediation of proliferative effects of BDNF and NT-3 is presented. Both of these neurotrophins elicited a fast and transient hydrolysis of labeled GPI, approximately 60% in 30 sec. The dose-response profile of GPI hydrolysis overlaps the neurotrophin-induced cell proliferation response profile. Anti-IPG antibodies were able to block the growth-promoting effects of BDNF and NT-3. Anti-IPG antibodies immunoprecipitated a CVG-endogenous IPG, induced upon BDNF treatment, which exhibited proliferative stimulating properties. Both BDNF and NT-3 are proposed as potential candidates for regulation of growth during CVG development, with this mitogenic effect being mediated by the GPI/IPG signaling system.

The use of cochlear implants (CIs) is on the rise for patients with vestibular schwannoma (VS). Besides CI following tumor resection, new scenarios such as implantation in observed and/or irradiated tumors are becoming increasingly common. A significant emerging trend is the need of intraoperative evaluation of the functionality of the cochlear nerve in order to decide if a CI would be placed. The purpose of this paper is to explore the experience of a tertiary center with the application of the Auditory Nerve Test System (ANTS) in various scenarios regarding VS patients. The results are compared to that of the studies that have previously used the ANTS in this condition. Patients with unilateral or bilateral VS (NF2) who were evaluated with the ANTS prior to considering CI in a tertiary center between 2021 and 2023 were analyzed. The presence of a robust wave V was chosen to define a positive electrical auditory brainstem response (EABR). Two patients underwent promontory stimulation (PromStim) EABR previous to ANTS evaluation. Seven patients, 2 NF-2 and 5 with sporadic VS were included. The initial scenario was simultaneous translabyrinthine (TL) tumor resection and CI in 3 cases while a CI placement without tumor resection was planned in 4 cases. The ANTS was positive in 4 cases, negative in 2 cases, and uncertain in one case. Two patients underwent simultaneous TL and CI, 1 patient simultaneous TL and auditory brainstem implant, 3 patients posterior tympanotomy with CI, and 1 patient had no implant placement. In the 5 patients undergoing CI, sound detection was present. There was a good correlation between the PromStim and ANTS EABR. The literature research yielded 35 patients with complete information about EABR response. There was one false negative and one false positive case; that is, the 28 implanted cases with a present wave V following tumor resection had some degree of auditory perception in all but one case. The ANTS is a useful intraoperative tool to asses CI candidacy in VS patients undergoing observation, irradiation or surgery. A positive strongly predicts at least sound detection with the CI.

Summary Background Previous studies indicate that murine models are useful tools for studying the allergic diseases, including certain aspects of bronchial asthma such as cellular tissue inflammation and pulmonary function. Objective To develop an experimental model of allergic lung inflammation based on a relevant human allergen, olive pollen, and to establish the immunological, cellular and functional airway features of the allergic response in this model. Methods Induction of systemic allergic response was achieved by the subcutaneous administration of Olea europaea extract in BALB/c mice. Olea ‐specific Igs (IgG1, IgG2a and IgE) and cytokines from splenocyte cultures IL‐4, IL‐5 IL‐10, IL‐13 and IFN‐γ were measured. Allergic airway response was generated by transnasal instillation of the allergens. Airway responsiveness was monitored by non‐invasive methacholine inhalation challenge. Lungs were paraffin embedded and histologically analysed. Apoptosis was studied by the TUNEL technique in the lung tissue and through cell cycle analysis by flow cytometry in splenocytes. Results Our results demonstrate that Olea ‐sensitized mice develop a specific allergic antibody (IgG1 and IgE) and cytokine (IL‐4, IL‐5, IL‐10 and IL‐13) response. After transnasal Olea instillation, they show inflammatory infiltration of lung tissue, mucus secretion and non‐specific hyper‐responsiveness in the airway. Concomitantly, differences in the rate of apoptosis are observed in the lung cells as well as a significant reduction of spontaneous apoptosis in the splenocytes of allergic mice. Conclusion We present a novel animal model of olive pollen‐allergic disease. This model presents traits associated with human allergic asthma and could be an interesting tool in the study of underlying molecular mechanisms and in exploring the therapeutic approaches to this disease.

To assess the validity of inducing ototoxicity in rats by applying a sponge soaked in kanamycin and furosemide on the round window.Basic, randomized, nonblind experimental study.Animal models of cochlear damage and reliable methods of local drug delivery are fundamental to study hearing loss and to design new therapies.Four experimental groups of six Wistar rats with different methods of drug administration were used: (1) injection of subcutaneous kanamycin (400 mg/kg) and intravenous furosemide (100 mg/kg); (2) local application of a sponge soaked in saline close to the round window; (3) animals for which the sponge was soaked in a solution containing kanamycin (200 mg/mL) and furosemide (50 mg/mL); and (4) sham-operated rats. The tympanic bulla was exposed using a ventral approach, and a bullostomy was performed to visualize the round window membrane. Cochlear function was assessed by measuring the auditory brainstem response, and hearing thresholds in response to click and tone burst stimuli were determined as peak and interpeak latencies. At the end of the study, cochlear histology was analyzed.Systemic administration of kanamycin and furosemide induced profound hearing loss and severe hair cell damage. Local delivery of these ototoxic drugs caused comparable damage but avoided the systemic side effects of the drug. Sham-operated and saline control animals did not experience functional alterations.Situating a sponge soaked in kanamycin and furosemide on the round window membrane through the ventral approach is a reliable method to provoke local ototoxicity in rats.

ABSTRACT Jun transcription factors have been implicated in the regulation of cell proliferation, differentiation and apoptosis. We have investigated the relationship between Jun expression and cell death in the developing chicken inner ear. c-jun and junD transcripts were expressed in the epithelium of the otic placode and otic vesicle. c-jun expression was restricted to the dorsal area of the otic pit (stages 14-17), dorsal otic vesicle and cochleo-vestibular ganglion (stages 18-20). junD expression was transient and occurred in the dorsal and upper medial aspects of the otic pit and otic cup, but it was down-regulated in the otic vesicle. A parallel TUNEL analysis revealed that expression of c-jun co-located within areas of intense apoptosis. Furthermore, phosphorylation of c-Jun at serine-63 by Jun amino-terminal-kinases was detected in the dorsal otic pit, otic vesicle and cochleo-vestibular ganglion. c-Jun protein exhibited DNA binding activity, as assessed by gel mobility shift assays. The association between c-Jun and apoptosis was further demonstrated by studying nerve growth factor-induced apoptosis in cultured otic vesicles. Nerve growth factor-induced cell death and c-Jun phosphorylation that were suppressed by insulin-like growth factor-I and by viral-mediated overexpression of Raf, which had survival effects. In conclusion, the precise regulation of the expression and activity of Jun proteins in the otic primordium suggests that it may operate as a fundamental mechanism during organogenesis.

S-adenosyl-L-methionine (SAM) is synthesized by methionine adenosyltransferases (MATs). Ablation of the liver-specificMAT1A gene results in liver neoplasia and sensitivity to oxidant injury.Here we show that acidic sphingomyelinase (ASMase) mediates the downregulation of MAT1A by TNF-α α.The levels of MAT1A mRNA as well as MAT I/III protein decreased in cultured rat hepatocytes by in situ generation of ceramide from exogenous human placenta ASMase.Hepatocytes lacking the ASMase gene (ASMase -/-) were insensitive to TNF-α α but were responsive to exogenous ASMase-induced downregulation of MAT1A.In an in vivo model of lethal hepatitis by TNF-α α, depletion of SAM preceded activation of caspases 8 and 3, massive liver damage, and death of the mice.In contrast, minimal hepatic SAM depletion, caspase activation, and liver damage were seen in ASMase -/-mice.Moreover, therapeutic treatment with SAM abrogated caspase activation and liver injury, thus rescuing ASMase +/+ mice from TNF-α α-induced lethality.Thus, we have demonstrated a new role for ASMase in TNF-α α-induced liver failure through downregulation of MAT1A, and maintenance of SAM may be useful in the treatment of acute and chronic liver diseases.Nonstandard abbreviations used: acidic sphingomyelinase (ASMase); alanine aminotransferase (ALT); Bacillus cereus sphingomyelinase (bNSMase); D-(+)-galactosamine (Gal); D-threo-1-phenyl-2-decanoylamino-3-morpholino-propanol HCl (D-threo-PDMP); ganglioside GD3 (GD3); ganglioside GM3 (GM3); glucosylceramide synthase (GCS); γ-glutamylcysteine synthetase (γ-GCS); glutathione (GSH); human placenta sphingomyelinase (hASMase); L-buthionine sulfoximine (BSO); liver-specific methionine adenosyltransferase (MAT1A); mannose 6-phosphate (M 6-P); methionine adenosyltransferase (MAT); N-acetylcysteine (NAC); neutral sphingomyelinase (NSMase); S-adenosyl-L-homocysteine (SAH); S-adenosyl-L-methionine (SAM); sphingomyelinase (SMase).

EDITORIAL article Front. Neurol., 09 February 2021 | https://doi.org/10.3389/fneur.2021.635359

Dear Editor, Cis-diammine-dichloroplatinum[II] (cisplatin) is a potent and widely used chemotherapeutic agent with significant efficacy against several forms of cancer in adults and children. Unfortunately, cisplatin has multiple adverse effects and causes irreversible hearing loss (HL) in a large proportion of patients, which severely impacts their quality of life.1 Here, we confirm that treatment with LPT99, a small molecule inhibitor of the apoptosome, preserves hearing levels in a rat model of cisplatin-induced HL and prevents apoptosis in cisplatin-exposed HEI-OC1 cells. These data are encouraging and support the potential of LPT99 for the prevention of the secondary effects of cisplatin for HL. Cisplatin primarily induces damage to outer (OHC) and inner hair cells and to spiral ganglion neurons, and long-term retention of cisplatin in the cochlea contributes to its irreversible ototoxic effects.2 Once inside the cell, cisplatin forms a highly reactive complex that induces DNA damage, activates death receptor pathways, and causes mitochondrial dysfunction, triggering reactive oxygen species generation, oxidative stress, and cytochrome c release. Cytochrome c binds with apoptotic protease activating factor 1 (APAF-1) forming the apoptosome, which recruits and activates caspase-9 and -3 to execute apoptosis3 (Figure 1A). Antioxidants and antiapoptotic drugs have been tested as otoprotectants in experimental models and clinical trials4 (Figure 1A), but their lack of specificity and efficacy are major limitations and none are currently approved for the treatment of cisplatin-induced HL. Accordingly, more effective agents are urgently needed, preferably formulated for local (intratympanic) administration to avoid interfering with the antineoplastic activity of cisplatin. As a central component of the apoptosome and a regulator of apoptosis, APAF-1 is a key pharmacological target that has been validated in experimental models.5 Orzáez et al.6 described a series of 2,5-piperazinedione derivatives (Patent US9040701B2) with APAF-1 inhibitory activity through binding to the APAF-1 caspase recruitment and nucleotide-binding oligomerization domains, which impair the conformational change required for its oligomerization and, consequently, inhibit the activation of procaspase-9 and the induction of apoptosis. These molecules attenuated cisplatin-induced apoptosis in HEI-OC1 cells and the loss of neuromasts in zebrafish, demonstrating that APAF-1 inhibition is effective against cisplatin-associated ototoxicity.6 We investigated the efficacy of LPT99 (Patent US10561736B1), a second-generation derivative with similar APAF-1 inhibitory activity but better pharmacological properties, in preventing cisplatin-induced ototoxicity using in vitro and in vivo models. The addition of 1 μM LPT99 to cisplatin-treated HEI-OC1 cell cultures7 significantly improved cell survival (p < 0.05) across the cisplatin concentration ranging from 1 to 4 μg/ml (Figure 1B, a). Flow cytometry analysis of annexin V/propidium iodide staining indicated that LPT99 alone did not trigger apoptosis, but significantly reduced the percentage of total apoptotic cells (early and late apoptosis) in co-treated cultures as compared with cisplatin alone (p < 0.001) (Figure 1B, b). Activated caspase-3 levels were also lower in cells co-treated with cisplatin and LPT99 (Figure 1B, c and d). LPT99 was formulated in cyclodextrin and Poloxamer 407 hydrogels to achieve sustained delivery, and was administered intratympanically8 in rats. Dosing of LPT99/cyclodextrin (50–200 μM) provided high LPT99 cochlear levels (Figure 2A, a) and had no effect on hearing thresholds even 30 days after administration (Figure 2A, b). Similarly, dosing of LPT99/Poloxamer 407 (100–797 μM) reached the cochlea, was detectable up to 14 days after injection, and had no effect on hearing, supporting its tolerability at the tested doses (Figure 2B). LPT99 was undetectable in plasma at all-time points and doses studied (quantification limit 5 ng/ml). We evaluated the efficacy of LPT99 by monitoring the auditory brainstem response (ABR) before and 3 days after bilateral intratympanic administration of LPT99 in cyclodextrin (Figure 2A, c) or in Poloxamer 407 (Figures 2C and 3) and intraperitoneal infusion of cisplatin (10 mg/kg). Analysis of the untreated group revealed that 86% of rats exhibited HL following cisplatin administration, whereas normal hearing was observed in 50%, 58%, and 70% of rats in the groups co-treated with 50, 100, or 478 μM LPT99/Poloxamer 407, respectively (Figure 2C). LPT99 treatment also notably reduced the percentage of animals with moderate and profound HL (Figure 2C). Co-administration of LPT99 reduced the cisplatin-induced threshold shifts for clicks (∼50% reduction) and pure tones (∼60%, 45%, and 35% reduction for 8, 16, and 28–40 kHz). Significant dose-dependent differences were found in threshold shifts between LPT99/Poloxamer 407-treated and nontreated rats, with the best protection profile observed at a dose between 100 μM and 478 μM LPT99 (Figure 3A and B). Cisplatin-induced changes in the latency and amplitude of the ABR waves were also attenuated by LPT99 (Figure 3C and D). The LPT99 dosing scheme improved hearing thresholds in a range similar to that reported for other otoprotective drugs, such as a p53 inhibitor,9 and improved upon those reported for the intracochlear perfusion of inhibitors of JNK, caspases-3, 9, and 8 and cathepsin B.10 The protective effects of LPT99/Poloxamer 407 were further confirmed by assessing cochlear cytoarchitecture (Figure 4). Cisplatin is known to induce substantial OHC loss in the basal and middle turns. Rats co-treated with LPT99 showed better preservation of the organ of Corti and spiral ganglion (Figure 4A–F, J, and L). The protective effect of LPT99 against cisplatin-induced OHC loss was further confirmed by the preserved E-cadherin and F-actin expression (Figure 4G, K, M, and Q) and the higher expression levels of prestin, a motor OHC protein (Figure 4H). LPT99-co-treated cochleae also showed less dilatation of strial microvasculature, particularly in the middle turn and exhibited higher levels of Kir4.1, a marker of intermediate cells (Figure 4I and O). Because the stria vascularis is the entry point of cisplatin into the cochlea and a long-term accumulation site, it has emerged as an important target to prevent cisplatin ototoxicity.2 Further studies are needed to demonstrate a direct effect of LPT99 on strial cells. In conclusion, LPT99 exerts dose-dependent protective effects on auditory function and cochlear cytoarchitecture in cisplatin-treated rats. LPT99 also protects HEI-OC1 cells against cisplatin-induced apoptosis, although further mechanistic studies will be needed to fully establish the participation of APAF-1. Overall, our findings highlight the role of APAF-1 as a pharmacological target for new drugs and point to LPT99 as a potential prophylactic local treatment for cisplatin-induced HL. We thank our colleagues of the Neurobiology of Hearing Group for critical comments and sharing unpublished information. We thank the Non-Invasive Neurofunctional Evaluation and Genomic facilities (Institute for Biomedical Research "Alberto Sols" CSIC-UAM) and the Histology facility (National Biotechnology Center, CSIC) for technical support. This work was supported by a SPIRALTH-CIBERER ER17PE12 grant to SMC. SMC and LRR hold CIBERER contracts. BC, AMC, and JMB contracts were supported in part by projects from FP7-PEOPLE-2013-IAPP-TARGEAR and from MINECO/FEDER SAF2017-86107-R to IVN. All authors read and approved the final manuscript. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. SMC has received research support from Spiral Therapeutics and IVN has received compensation for consulting. The remaining authors declare that they have no significant competing financial, professional, or personal interests that might have influenced the performance or presentation of the work described in this manuscript. Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER-SPIRALTH ER17PE12); Seventh Framework Programme, People: Marie-Curie Actions, Industry-Academia Partnerships (FP7-PEOPLE-2013-IAPP-TARGEAR); European Regional Development Fund, Spanish Ministerio de Economía y Competitividad (SAF2017-86107-R) Participated in research design: Silvia Murillo-Cuesta and Isabel Varela-Nieto; conducted experiments: Silvia Murillo-Cuesta, Blanca Cervantes, Lourdes Rodríguez de la Rosa, Jose M. Bermúdez-Muñoz, Julio Contreras, and Adelaida M. Celaya; performed data analysis: Silvia Murillo-Cuesta, Blanca Cervantes, Lourdes Rodríguez de la Rosa, Adelaida M. Celaya, Isabel Sánchez-Pérez, and Isabel Varela-Nieto; wrote or contributed to the writing of the manuscript: Silvia Murillo-Cuesta, Isabel Sánchez-Pérez, and Isabel Varela-Nieto.

The verterbrate inner ear is an excellent model system to study signalling mechanisms in embryonic development. During the last years, insulin-like growth factor-I (IGF-I) has attracted attention in relation to the regulation of inner ear ontogenesis. IGF-I and its high-affinity tyrosine-kinase receptor are expressed during early stages of inner ear development. IGF-I is a powerful mitogen for the otic vesicle, where it stimulates cell-division and mitogenic signalling cascades. Later in development, IGF-I also promotes survival and neurogenesis of the otic neurones in the cochleovestibular ganglion (CVG). The actions of IGF-I are associated with the generation of lipidic messengers and the activation of Raf kinase, which results in the rapid induction of the expression of the proliferative cell nuclear antigen (PCNA) and the nuclear proto-oncogenes c-fos and c-jun. Regulation of organogenesis involves a dynamic balance of the mechanisms regulating cell division, differentiation and death. A model is proposed where this balance is the consequence of the action of IGF-I and NGF, which converge in Raf activation or suppression. The combinatorial expression of jun and Fos family members in particular domains of the otic vesicle would be the final result of such cascade. Some of these mechanisms may be also implicated in otic regeneration.

The ability of an inositol phosphate-glycan (IPG) to mimic the effects of insulin on regulation of the expression of specific mRNAs was studied in isolated hepatocytes from normal and diabetic rats. Incubation of normal liver cells with IPG (10 μm) during 90 min produced a 5-fold decrease in phosphoenolpyruvate carboxykinase (PEPCK) mRNA levels, which had been previously increased about 10-fold by incubation with 8-bromo-cAMP (0.1 mm). The effect of IPG was dose dependent and could not be reproduced by galactose, glucosamine, or myo-inositol. IPG reduction of PEPCK mRNA is primarily due to a decrease in the rate of transcription of the gene, as judged by nuclear run-on transcription experiments performed in rat hepatoma H4IIE cells. In hepatocytes isolated from diabetic rats, treatment with 5 μm IPG for 15 min caused a 4-fold induction in the expression of α2-microglobulin mRNA concomitantly with a 2.5-fold decrease in the level of PEPCK mRNA. Cleavage of IPG with nitrous acid abolished both the increase and the decrease in specific mRNAs levels. Glycosyl-phosphatidylinositol, the lipid precursor of IPG, did not modify either PEPCK or α2-microglobulin mRNA levels. These data indicate that both positive and negative effects of insulin on the regulation of gene expression are mimicked by IPG.

Betaine-homocysteine S-methyltransferases (BHMTs) are methionine cycle enzymes that remethylate homocysteine; hence, their malfunction leads to hyperhomocysteinemia. Epidemiologic and experimental studies have revealed a correlation between hyperhomocysteinemia and hearing loss. 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 before and following exposure to noise. Also, we measured cochlear cytoarchitecture, gene expression by RNA-arrays and quantitative RT-PCR, 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, outer hair cell loss, 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 up-regulation could carry out a compensatory role in cochlear protection against noise injury in the absence of BHMT.-Partearroyo, T., Murillo-Cuesta, S., Vallecillo, N., Bermúdez-Muñoz, J. M., Rodríguez-de la Rosa, L., Mandruzzato, G., Celaya, A. M., Zeisel, S. H., Pajares, M. A., Varela-Moreiras, G., Varela-Nieto, I. Betaine-homocysteine S-methyltransferase deficiency causes increased susceptibility to noise-induced hearing loss associated with plasma hyperhomocysteinemia.

Abstract 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), among other deleterious agents, which elicits a strong inflammatory response in the inner ear leading to irreversible otic damage. Prolonged administration of systemic corticosteroids is the available treatment, which 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 ll1lr , 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.

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

Molecular conservation and novelties in vertebrate ear development Use of mouse genetics for studying inner ear development Formation of the outer and middle ear, molecular mechanisms Molecular basis of inner ear induction FGF signaling in ear development and innervation The roles of retinoic acid during inner ear development Hair cell development in higher vertebrates Growth factors and early development of otic neurons The cell cycle and the development and the regeneration of hair cells

Signal transduction through the hydrolysis of glycosyl-phosphatidylinositol (GPI) leading to the release of the water-soluble inositol phosphoglycan (IPG) molecules has been demonstrated to be important for mediating some of the actions of insulin and insulin-like growth factor-I (IGF-I). In the present study, GPI from grass pea (Lathyrus sativus) seeds has been purified and partially characterized on the basis of its chromatographic properties and its compositional analysis. The results indicate that it shows similarities to GPI previously isolated from other sources such as rat liver. IPG was generated from L. sativus seed GPI by hydrolysis with a GPI-specific phospholipase D (GPI-PLD). This IPG inhibited protein kinase A (PKA) in an in vitro assay, caused cell proliferation in ex-planted cochleovestibular ganglia (CVG), and decreased 8-Br-cAMP-induced phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression in cultured hepatoma cells. Our data indicate that L. sativus seed IPG possess insulin-mimetic activities. This may explain why L. sativus seeds have been used in some traditional medicines to ameliorate diabetic symptoms.

Nitrones are potent antioxidant molecules able to reduce oxidative stress by trapping reactive oxygen and nitrogen species. The antioxidant potential of nitrones has been extensively tested in multiple models of human diseases. Sensorineural hearing loss has a heterogeneous etiology, genetic alterations, aging, toxins or exposure to noise can cause damage to hair cells at the organ of Corti, the hearing receptor. Noxious stimuli share a battery of common mechanisms by which they cause hair cell injury, including oxidative stress, the generation of free radicals and redox imbalance. Therefore, targeting oxidative stress-mediated hearing loss has been the subject of much attention. Here we review the chemistry of nitrones, the existing literature on their use as antioxidants and the general state of the art of antioxidant treatments for hearing loss.

Organogenesis involves a dynamic balance of the mechanisms regulating cell division, differentiation and death. The development of the chicken embryo inner ear offers a well-characterised model at the morphological level to study which signals are implicated in the modulation of cellular activation and commitment. The early developmental decisions that control the origin of the inner ear elements are just beginning to be identified by complementary in vivo and in vitro studies. Insulin-like growth factor-I (IGF-I) and nerve growth factor (NGF) are among the best characterised diffusible factors acting during inner ear development. Although the cellular actions of these factors are beginning to be understood, the signalling pathways triggered by them still remain largely unknown. In this context, viral vehicles can be used to deliver genes and then analyse their functional roles during inner ear development. A model is proposed where the actions of IGF-I and NGF contribute to the combinatorial expression of Jun and Fos family members in particular domains of the otic vesicle. Some of these mechanisms may be also implicated in otic regeneration.

Insulin-like growth factor (IGF)-I is a widely expressed growth factor with diverse effects on many tissues throughout development and in adult life. The purpose of this work is to provide detailed an

We present two minimally invasive microsurgical techniques in rodents for specific drug delivery into the middle ear so that it may reach the inner ear. The first procedure consists of perforation of the tympanic bulla, termed bullostomy; the second one is a transtympanic injection. Both emulate human clinical intratympanic procedures. Chitosan-glycerophosphate (CGP) and Ringer´s Lactate buffer (RL) were used as biocompatible vehicles for local drug delivery. CGP is a nontoxic biodegradable polymer widely used in pharmaceutical applications. It is a viscous liquid at RT but it congeals to a semi solid phase at body temperature. RL is an isotonic solution used for intravenous administrations in humans. A small volume of this vehicle is precisely placed on the Round Window (RW) niche by means of a bullostomy. A transtympanic injection fills the middle ear and allows less control but broader access to the inner ear. The safety profiles of both techniques were studied and compared by using functional and morphological tests. Hearing was evaluated by registering the Auditory Brainstem Response (ABR) before and several times after microsurgery. The cytoarchitecture and preservation level of cochlear structures were studied by conventional histological techniques in paraformaldehyde-fixed and decalcified cochlear samples. In parallel, unfixed cochlear samples were taken and immediately frozen to analyze gene expression profiles of inflammatory markers by quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR). Both procedures are suitable as drug delivery methods into the mouse middle ear, although transtympanic injection proved to be less invasive compared to bullostomy.

The involvement of the glycosylphosphatidylinositol/ inositol phosphoglycan (gly-PtdIns/IPG) system in the stimulation of macromolecular syntheses in human fibroblasts has been investigated. The study demonstrates that an insulin sensitive gly-PtdIns/IPG system is present in human fibroblasts, that IPG can significantly stimulate DNA, RNA, and protein synthesis, and that the action of insulin on DNA synthesis as well as that of IPG can be significantly reduced by a specific anti-IPG antibody. These results strongly support the hypothesis that the gly-PtdIns/IPG system is involved in the signal transduction pathway leading to the stimulation of Cell proliferation.

Insulin-like growth factor I (IGF-I) signaling is mediated by insulin receptor substrate proteins (IRS). In turn, protein tyrosine phosphatase 1B (PTP1B) dephosphorylates and inactivates insulin and IGF-I receptors. IRS2-deficient mice present developmental defects in the nervous system, altered hepatic insulin signalling b-cell failure and develop type 2-like diabetes. IGF1 gene mutations cause syndromic sensorineural hearing loss in men and mice. However, the involvement of IRS2 and PTP1B, two IGF-1 downstream signaling mediators, in hearing onset and loss has not been studied. We have studied here, the hearing function and cochlear morphology of IRS2 null mice and the impact of PTP1B deficiency. Auditory brainstem responses and cochlear morphology of systemic Irs2−/−Ptpn1+/+, Irs2+/+Ptpn1-/and Irs2−/−Ptpn1−/− mice were studied at different postnatal ages. The results indicated that Irs2−/−Ptpn1+/+ mice present a profound congenital sensorineural deafness before the onset of diabetes and altered cochlear morphology compared to wild type mice. Simultaneous PTP1B deficiency in Irs2−/−Ptpn1−/− mice delays the onset of deafness. We show for the first time that IRS2 is essential for hearing and that PTP1B inhibition may be useful for the treatment of deafness associated to hyperglycemia and type 2-diabetes. We warmly thank the support of our colleagues Carlos Avendano, Deborah Burks and Lupe Camarero.

EDITORIAL article Front. Cell. Neurosci., 30 March 2022Sec. Cellular Neurophysiology https://doi.org/10.3389/fncel.2022.882417

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Age-related hearing loss (ARHL) is the most common sensory deficit in the elderly. The disease has a multifactorial etiology with both environmental and genetic factors involved being largely unknown. SLC7A8/SLC3A2 heterodimer is a neutral amino acid exchanger. Here, we demonstrated that SLC7A8 is expressed in the mouse inner ear and that its ablation resulted in ARHL, due to the damage of different cochlear structures. These findings make SLC7A8 transporter a strong candidate for ARHL in humans. Thus, a screening of a cohort of ARHL patients and controls was carried out revealing several variants in SLC7A8, whose role was further investigated by in vitro functional studies. Significant decreases in SLC7A8 transport activity was detected for patient’s variants (p.Val302Ile, p.Arg418His, p.Thr402Met and p.Val460Glu) further supporting a causative role for SLC7A8 in ARHL. Moreover, our preliminary data suggest that a relevant proportion of ARHL cases could be explained by SLC7A8 mutations. https://doi.org/10.7554/eLife.31511.001 eLife digest Age-related hearing loss affects about one in three individuals between the ages of 65 and 74. The first symptom is difficulty hearing high-pitched sounds like children’s voices. The disease starts gradually and worsens over time. Changes in the ear, the nerve that connects it to the brain, or the brain itself can cause hearing loss. Sometimes all three play a role. Genetics, exposure to noise, disease, and aging may all contribute. The condition is so complex it is difficult for scientists to pinpoint a primary suspect or develop treatments. Now, Guarch, Font-Llitjós et al. show that errors in a protein called SLC7A8 cause age-related hearing loss in mice and humans. The SLC7A8 protein acts like a door that allows amino acids – the building blocks of proteins – to enter or leave a cell. This door is blocked in mice lacking SLC7A8 and damage occurs in the part of their inner ear responsible for hearing. As a result, the animals lose their hearing. Next, Guarch, Font-Llitjós et al. scanned the genomes of 147 people from isolated villages in Italy for mutations in the gene for SLC7A8. The people also underwent hearing tests. Mutations in the gene for SLC7A8 that partially block the door and prevent the flow of amino acids were found in people with hearing loss. Some mutations in SLC7A8 that allow the door to stay open where found in people who could hear. The experiments suggest that certain mutations in the gene for SLC7A8 are likely an inherited cause of age-related hearing loss. It is possible that other proteins that control the flow of amino acids into or out of cells also may play a role in hearing. More studies are needed to see if it is possible to fix errors in the SLC7A8 protein to delay or restore the hearing loss. https://doi.org/10.7554/eLife.31511.002 Introduction Age-related hearing loss (ARHL) or presbycusis is one of the most prevalent chronic medical conditions associated with aging. Indeed, more than 30% of people aged over 65 years suffer ARHL (Gates and Mills, 2005; Huang and Tang, 2010; Van Eyken et al., 2007). Clinically, ARHL is defined as a progressive bilateral sensorineural impairment of hearing in high sound frequencies mainly caused by a mixture of 3 pathological changes: loss of the hair cells of the organ of Corti (sensory), atrophy of the stria vascularis (metabolic) and degeneration of spiral ganglion neurons (SGN), as well as the central auditory pathway (neural) (Gates and Mills, 2005; Schuknecht, 1955; Yamasoba et al., 2013). ARHL has a complex multifactorial etiology with both genetic and environmental factors contributing (Cruickshanks et al., 2010; Christensen et al., 2001). Although most people lose hearing acuity with age, it has been demonstrated that genetic heritability affects the susceptibility, onset and severity of ARHL (Wingfield et al., 2007; Cruickshanks et al., 2001; Gates et al., 1999; Karlsson et al., 1997; Cruickshanks et al., 1998). Unfortunately, the complexity of the pathology coupled with highly variable nature of the environmental factors, which cause cumulative effects, increases the difficulty in identifying the genetic contributors underlying ARHL. Most of the findings from genome-wide association studies (GWAS) performed into adult hearing function could neither be replicated between populations, nor the functional validation of those candidates be confirmed (Dawes and Payton, 2016). Mouse models, including inbred strains, have been essential for the identification of several defined loci that contribute to ARHL (Bowl and Dawson, 2015). SLC7A8/SLC3A2 is a Na+‐independent transporter of neutral amino acids that corresponds to system L also known as LAT2 (L-type Amino acid Transporter-2) (Pineda et al., 1999; Rossier et al., 1999; Oxender and Christensen, 1963). SLC7A8 is the catalytic subunit of the heterodimer and mediates obligatory exchange with 1:1 stoichiometry of all neutral amino acids, including the small ones (e.g. alanine, glycine, cysteine and serine), which are poor substrates for SLC7A5 (18), another exchanger with system L activity. Functional data indicate that the role of SLC7A8 is to equilibrate the relative concentrations of different amino acids across the plasma membrane instead of mediating their net uptake (Pineda et al., 1999; Meier et al., 2002; Verrey, 2003). The SLC7A8/SLC3A2 heterodimer is primarily expressed in renal proximal tubule, small intestine, blood-brain barrier and placenta, where it is thought to have a role in the flux of amino acids across cell barriers (Rossier et al., 1999; Bauch et al., 2003; Kanai and Endou, 2001; del Amo et al., 2008). So far, SLC7A8 research has been focused mainly on amino acid renal reabsorption. However, in vitro studies demonstrated that SLC7A8 could have a role in cystine efflux in epithelial cells and the in vivo deletion of Slc7a8 in a mouse model showed a moderate neutral aminoaciduria (Braun et al., 2011), suggesting compensation by other neutral amino acid transporters. Therefore, in order to better understand the physiology of SLC7A8, we generated null Slc7a8 knockout mice (Slc7a8−/−) (Font-LLitjós, 2009) and (Figure 1—figure supplement 1A). Here, we describe the detection of a hypoacusic phenotype in the Slc7a8−/− mouse model and demonstrate that novel loss-of-function SLC7A8 mutations constitute a primary cause in the development of ARHL in a cohort of elderly people from two isolated villages in Italy. Results Slc7a8 ablation causes ARHL SLC7A8 is highly expressed in the kidney, intestine and brain, and neither full-length nor truncated SLC7A8 protein were detected in membrane samples of Slc7a8−/− mice (Figure 1A). The Allen Brain Atlas (Allen Institute for Brain Science, 2004) localizes mouse brain SLC7A8 to the cortical subplate, cerebellum, thalamus and olfactory bulb. Our results showed that SLC7A8 protein was localized to the plasma membrane of neuronal axons in different brain regions such as, the choroid plexus, subfornical organ, cerebral cortex and hypothalamus by immunohistochemistry (Figure 1—figure supplement 2A). This specific localization in the brain pointed to the possibility that the absence of the transporter could potentially lead to neurological disorders. Behavioral screening showed that absence of SLC7A8 in mice does not affect either learning or memory (Figure 1—figure supplement 3). In contrast, a significant reduction in latency was observed in the rotarod acceleration test indicating impairment in motor coordination in Slc7a8−/− mice (Figure 1—figure supplement 3G). Reaffirming poorer motor coordination performance in the Slc7a8−/− mice, an increased exposure to shock on the treadmill was also observed (Figure 1—figure supplement 3B). Interestingly, a marked impairment was observed in the pre-pulse inhibition of acoustic startle response, which assesses the response to a high intensity acoustic stimulus (pulse) and its inhibition by a weaker pre-pulse. The response to a 120 dB single-pulse was significantly reduced in Slc7a8−/− mice (Figure 1B). The higher threshold required for responding to the acoustic stimulus in the PPI tests in Slc7a8−/− animals could potentially be indicative of a hearing impairment or to a defect in the stress response signaling. Figure 1 with 5 supplements see all Download asset Open asset Hearing phenotype of C57BL6/J-129Sv Slc7a8 knockout mice. (A) Representative image of western bloting of total membranes from kidney, brain and intestine of wild-type (+/+) and Slc7a8 knock out (-/-) mice in the absence (-) or presence (+) of 100 mM dithiothreitol reducing agent (DTT) of three independent biological samples for both sexes (male and female). Protein (50 μg) were loaded in 7% acrylamide SDS-PAGE gel. Molecular mass standard (KDa) are indicated. Red arrows point SLC7A8/CD98hc heterodimer band as well as the light subunit SLC7A8. Upper panel: Rabbit anti-SLC7A8. Bottom panel: Mouse anti-βactin. (B) Pre-Pulse Inhibition of the acoustic startle response (PPI). Mean and SEM are represented. Pulse: 120 dB single pulse. Pre-pulse inhibition test: six different pre-pulse intensities (70 to 90 dB) in pseudo random order with 15 s inter-trial intervals. Wild type (white circles, n = 19) and Slc7a8 −/− (blue circles, n = 15) from 4- to 7-month-old are represented. Significant differences were determined using paired Student’s t-test, ***p<0.001 (C–F) Hearing phenotype in wild-type (Slc7a8+/+, white, n = 11), heterozygous (Slc7a8+/−, green, n = 12) and knockout (Slc7a8−/−, blue, n = 14) mice, grouped by age (4–6 and 7–13 month old). (C,D) Auditory Brainstem Response (ABR) threshold in response to click, expressed as mean ±standard error (C), individual value (scatter plot, (D) and median (boxplot, (D). The significance of the differences was evaluated using ANOVA test, *p<0.05, **p<0.01 (Slc7a8−/− versus Slc7a8+/+) and # p<0.05 (Slc7a8−/− versus Slc7a8+/−). (E) Pie plot showing the percentage of normal hearing (all thresholds <45 dB SPL, white) mice and mice with mild (at least two tone burst threshold >45 dB SPL, orange) and severe (at least two tone burst threshold >60 dB SPL, red) hearing loss (HL), within each genotype and age group. (F) ABR thresholds in response to click and tone burst stimuli (8, 16, 24, 32 and 40 kHz) in mice from three genotypes separated by age group and hearing phenotype (normal hearing or hearing loss). Significant differences were determined using ANOVA test, *p<0.05, **p<0.01, ***p<0.001 (hearing impaired Slc7a8−/− versus normal hearing Slc7a8+/+) and # p<0.05 (hearing impaired Slc7a8−/− versus Slc7a8+/−). https://doi.org/10.7554/eLife.31511.003 Response to stress is modulated by the hypothalamic-pituitary-adrenal axis via the release of corticosterone from the adrenal cortex (Smith and Vale, 2006). As SLC7A8 is expressed in the murine pituitary gland (Figure 1—figure supplement 2A and S3H), plasma corticosterone levels under stressing conditions were analyzed. No differences were observed in corticosterone levels at either basal conditions, nor under restraint stress in the Slc7a8−/− group, indicating a normal stress response in the absence of SLC7A8 (Figure 1—figure supplement 3I). Thus, a hearing impairment in Slc7a8−/− animals was considered the most probable cause of the differences observed in the acoustic startle response test (Figure 1B). The impact of the ablation of SLC7A8 on the auditory system was tested initially on mice with a mixed C57BL6/J‐129Sv genetic background. Auditory brainstem response (ABR) recording, which evaluates the functional integrity of the auditory system, was performed in Slc7a8−/− mice. Reinforcing our hypothesis, adult 4- to 6-month-old Slc7a8−/− mice showed significantly higher (p≤0.01) ABR thresholds in response to click stimulus, compared with age matched Slc7a8+/− and wild type mice, which maintain normal hearing thresholds (Figure 1C–E). The hearing loss observed in Slc7a8−/− mice affected the highest frequencies tested (20, 28 and 40 kHz) (Figure 1F). The analysis of latencies and amplitudes of the ABR waves in response to click stimuli, showed increased latency and decreased amplitude of wave I, but similar II-IV interpeak latency, in the Slc7a8−/− mice when compared with the other genotypes, pointing to a hypoacusis of peripheral origin without affectation of the central auditory pathway (Figure 1—figure supplement 4A to D). Mice were grouped according to genotype, age and ABR threshold level and descriptive statistics calculated, showing that the penetrance of the hearing phenotype in the Slc7a8−/− mice is incomplete (Figure 1D and E). Therefore, mice were classified according to their hearing loss (HL) phenotype, defining normal hearing when ABR thresholds for all frequencies were <45 dB SPL, mild phenotype when at least two thresholds were between 45 and 60 dB SPL and severe hypoacusis when at least two thresholds were >60 dB SPL. At 4–6 months of age, Slc7a8−/− mice showed either severe (37.5%) or mild (25%) hearing loss, whilst mice from the other genotypic groups did not show hearing loss (Figure 1E). Next we studied 7–13 month-old mice, 50% of Slc7a8−/− mice presented severe hypoacusis and the hearing loss spread to lower frequencies with age. Slc7a8−/− mice with hearing loss showed statistically significant differences in ABR parameters when compared to the other genotypes (Figure 1F). Moreover, 43% of Slc7a8+/− mice developed mild hearing loss at 7–13 months, whereas the age-matched wild-type mice maintained intact hearing indicating a predisposition toward hearing loss in aged Slc7a8+/−mice (Figure 1E). The onset and severity of ARHL is attributed to both environmental and genetic factors (Cruickshanks et al., 2010). As the environmental factors were well controlled in all the experiments, thus the phenotypic variability could be attributed as the consequence of individual genetic differences. Indeed, it has been described that several strains of inbred mice present a predisposition to suffer ARHL dependent on multiple genetic factors (Kane et al., 2012; Murillo-Cuesta et al., 2010). Here, the hearing loss phenotype was confirmed in a second mouse strain, the inbread C57BL6/J genetic background (Figure 1—figure supplement 5). Additionally, longitudinal study of Slc7a8−/− mice into the inbred C57BL6/J genetic background showed higher penetrance than the mixed background throughout the ages studied (Figure 2—figure supplement 2). Localization and quantification of SLC7A8 in the inner ear The presence of SLC7A8 has previously been reported in the mouse cochlea (Yang et al., 2011; Uetsuka et al., 2015; Sharlin et al., 2011), and specifically localized to the stria vascularis by liquid chromatography tandem mass spectrophotometry and by Western blotting (Uetsuka et al., 2015). Here, SLC7A8 was detected in wild-type mouse cochlea by immunofluorescence supporting its localization to the spiral ligament and spiral limbus from the basal to the apical regions of the cochlea (Figure 2A and B). SLC7A8 immunolabeling was not observed in the stria vascularis. We observed an intense expression of SLC7A8 in the spiral ligament surrounding the stria indicating that the SLC7A8 epitope (Figure 1—figure supplement 1B) is either hidden or absent in the stria vascularis. Quantification of SLC7A8 expression in the cochlea showed half a dose of the transporter in the Slc7a8+/−than in wild-type mice, and its ablation in Slc7a8−/− mice (Figure 2C). A closer study of SLC7A8 immunofluorescence showed that the transporter is also expressed in the spiral ganglia neurons area (SGN) (Figure 1—figure supplement 2B). Figure 2 with 2 supplements see all Download asset Open asset Immunolocalization of SLC7A8 in the mouse cochlea. (A) Representative photomicrographs of cryosections of the base of the cochlea showing immunodetection for SLC7A8 (green) and s100 (red); and staining for DAPI (blue) or phalloidin (white) of wild type (upper row) and Slc7a8−/− mice (lower row). Scale bar, 100 µm. (B) On the left overlay image of a wild-type section indicating cochlea areas. Scale bar, 100 µm. On the right schematic drawing of the adult scala media adapted from Sanchez-Calderon et al. (2010). BC, border cells; CC, Claudius's cells; DC, Deiter's cells; HC, Hensen's cells; IC, intermediate cells; IHC, inner hair cells; IPC, inner phalangeal cells; Li, spiral limbus; MB, Basilar Membrane; OHC, outer hair cells; PC, pillar cells; RM, Reisner's membrane; SG, spiral ganglion; SL, spiral ligament; SV, stria vascularis; TM, tectorial membrane. (C) Quantification of SLC7A8 expression. Intensity of SLC7A8 immunofluorescence was normalized per mm2. Mean ±SEM from quadruplicates for each section, taken from apex, middle and basal cochlear turns of 4 wild-type (black), 3 Slc7a8+/− (green) and 4 Slc7a8−/− (blue) young (4- to 7-month-old) mice. Open and closed circles represent individual mice from C57BL6/J-129Sv or C57BL6/J backgrounds, respectevely. Unpaired Student’s t-test statistical analysis, p-values: *,≤0.05; **,≤0.01 and ***,≤0.001. (D) Quantification of SLC7A8 protein expression in the apex, middle and basal cochlear turns normalized per nuclei of young (2 month-old) (open bars) and old (12 month-old) (black bars) wild-type CBA mice. Data (mean ±SEM) were obtained from four cochlear sections obtained from three mice per group. Unpaired Student’s t-test statistical analysis, p-value: *,≤0.05. https://doi.org/10.7554/eLife.31511.009 The early HL onset and the progressive ARHL phenotype observed in Slc7a8−/− and Slc7a8+/− mice respectively, prompted us to compare the expression of SLC7A8 in wild-type cochlea at different ages (Figure 1D). Immunofluorescence quantification of SLC7A8 intensity at 2- and 12 months of age showed expression in the young mice and increased presence of the transporter in the older mice (Figure 2D). In the same line, Slc7a8 mRNA quantification from cochlea extracts showed a progressive increased expression throughout mouse life (Figure 2—figure supplement 1A). Lack of Slc7a8 induced damage in the organ of Corti, spiral ganglion and stria vascularis The cytoarchitecture of the inner ear was studied by hematoxylin/eosin staining (Figure 3), immunofluorescence (Figure 4 and Figure 4—figure supplement 1) and mRNA detection of several cochlear markers (Figure 3D and Figure 2—figure supplement 1). Most of the structures of the cochlear duct, including spiral ligament, spiral limbus, tectorial and basilar membranes showed a normal gross cytoarchitecture in the Slc7a8−/− mice. In contrast, in the basal turns of the cochlea we observed that 3 out of 6 Slc7a8−/− mice evaluated showed complete loss of hair cells and flat epithelia, while only one Slc7a8−/− mouse showed intact epithelia in the organ of Corti (Figure 3A). Likewise, loss of cells in the spiral ganglia, especially in the basal regions of the cochlea, was observed (Figure 3A). Slc7a8−/− mice at 4 to 7 months of age presented ~50% of cell loss in the spiral ganglion compared with wild type mice (Figure 3B). Decreased number of cells in the ganglia significantly correlates with ABR threshold and HL phenotype (Figure 3—figure supplement 1and B). Concomitantly with the loss of hair cells and spiral ganglion (SG) nuclei in Slc7a8−/− mice, the messenger levels of cell type specific biomarkers, such as the potassium voltage-gated channels Kcnq2, Kcnq3 and Kcnq5, and the transporter Slc26a5, which are expressed in the organ of Corti and SG were down-regulated respectively (Figure 3D and Figure 2—figure supplement 2B). Figure 3 with 1 supplement see all Download asset Open asset Cytoarchitecture of the Slc7a8−/− mouse cochlea. (A) Hematoxylin and Eosin staining of the base of the cochlea. Representative photomicrographs taken from paraffin sections of wild-type and hipoacusic Slc7a8−/− mice. OC, Organ of Corti; SG, spiral ganglia region; and SL, spiral ligament. * Indicates loss of hair cells in the organ of Corti (first column), loss of neurons in the spiral ganglia (second column) and lower nuclei density in the spiral ligament (third column). Scale bar 100 μm. (B) Quantification of the number of neurons in the spiral ganglia (SG) in the basal turns of the cochlea. Y axis represents the mean nuclei quantification of 5 to 10 areas in SG. (C) Quantification of the number of nuclei in the spiral ligament (SL) of the basal turns of the cochlea by immunofluorescence using DAPI staining. For each sample, 12 overlaps of Z-stacks areas were used to quantify number of nuclei. Unpaired Student’s t-test statistical analysis: **, p≤0.01 (A to C) 4 wild-type (black), 3 Slc7a8+/− (green) and 4 Slc7a8−/− (blue) mice at 4 to 7-month-old are represented. Circles represent the average of the quadruplicate analysis performed in each mouse of C57BL6/J-129Sv (open) and C57BL6/J (filled) background. (D) Quantification of mRNA markers by RT-qPCR PCR. Cochlear gene expression of Slc26a5, Tbx18, Kcnq2 and Kcnq3 in the cochlea at 3-month-old and 7 months wild-type (white bars) and Slc7a8−/− (blue bars) C57BL6/J mice. Expression levels, normalized with Rplp0 gene expression, are represented as n-fold relative to control group. Values are presented as mean ±SEM of triplicates from pool samples of three mice per condition. Unpaired Student’s t-test statistical analysis, p-values: *p≤0.05; **p≤0.01; ***p≤0.001. https://doi.org/10.7554/eLife.31511.012 Figure 4 with 1 supplement see all Download asset Open asset Immunofluorescence of cochlear markers in the Slc7a8−/− mouse. (A) Representative photomicrographs of cryosections (10 μm) from the basal turn of the cochlea from wild type (1 and 4), Slc7a8+/− (2 and 5) and Slc7a8−/− (3 and 6) mice labeled for Kir4.1 (green), phalloidin (red) and DAPI (blue) (1 to 3), or for s100 (red), phalloidin (cyan) and DAPI (blue) (4 to 6). Scale bar, 100 µm. (B, C and D) Graph representing the quantification of Kir4.1, s100 and phalloidin (Pha) labeling intensity in the basal turn of the cochlea. Means ± SEM, normalized per mm2 of 4 wild type (black bars), 3 Slc7a8+/− (green bars) and 4 Slc7a8−/− (blue bars) young (4- to 7-month-old) mice are represented. Individual circles represent the average of the quadruplicate analysis of sections from each mice of either C57BL6/J-129Sv (open) or C57BL6/J (filled) backgrounds. Unpaired Student’s t-test statistical analysis, p-value: *,≤0.05. https://doi.org/10.7554/eLife.31511.014 Less densely packed cells in the spiral ligament were observed in Slc7a8−/− than in wild-type mice (Figure 3A). Reinforcing this observation, the expression of Kir4.1, a potassium channel highly expressed in stria vascularis cells (Ando and Takeuchi, 1999), was also dramatically reduced by 50% in Slc7a8−/− (Figure 4B and Figure 4—figure supplement 1A). Likewise, decreased expression of Kir4.1 marker correlates with HL phenotype (Figure 3—figure supplement 1C). Phalloidin labeling of actin fibers in the basal cells of the stria vascularis was also decreased 50% in the base of the cochlea (Figure 4C and Figure 4—figure supplement 1). SLC7A8 is abundantly expressed in fibrocytes of the spiral ligament and limbus (Figure 2), accordingly the number of fibrocytes in the spiral ligament decreased by 2/3 and 1/3 in the null and Slc7a8+/− mice, respectively (Figure 3C). Moreover, mice with severe HL phenotype showed 30% less number of fibrocytes in the spiral ligament (Figure 3—figure supplement 1D). The expression of the transcription factor Tbx18, essential for fibrocytes development and differentiation, was 50% less in Slc7a8−/− than in wild-type mouse cochleae (Figure 3D). In contrast, the expression of s100, fibrocyte types I and II marker, did not show significant differences (Figure 4D and Figure 4—figure supplement 1C). Mutations in SLC7A8 are associated with ARHL Once we associated mouse SLC7A8 transporter with deafness and identified it as a potential ARHL gene, screening for mutations in human populations was initiated. Whole genome sequencing (WGS) and audiogram test data obtained from 147 individuals from isolated villages in Italy were included in the study. The inclusion criteria were people 50 years old or older with an audiogram test done at high frequencies (Pure-tone audiometric PTA-H, 4 and 8 kHz). Individuals with pure-tone average for high frequencies (PTA-H) greater than or equal to 40 decibels hearing level (dB HL) were considered ARHL cases, whilst people with PTA-H less than 25 dB were considered as controls. A total of 66 cases suffering ARHL and 81 controls were selected. The gene-targeted studies conducted in this isolated cohort succeeded in detecting seven heterozygous missense variants (Table 1). Four of the variants: p.Val460Glu (V460E), p.Thr402Met (T402M), p.Val302Ile (V302I) and p.Arg418His (R418C) belong to ARHL cases (see Audiogram in Figure 5—figure supplement 1A) and other three: p.Arg8Pro (R8P), p.Ala94Thr (A94T) and p.Arg185Gln (R185L) to the control group (see Audiogram in Audiogram in Figure 5—figure supplement 1B). Table 1 SLC7A8 Humans mutations found in ARHL and controls individuals. https://doi.org/10.7554/eLife.31511.016 PhenotypeAgeSexChr. 14VariantConsequenceCodeFrequencyEsp6500siv21000 gCampionExACStudied cohortARHL75Female2359729014:23597290 A / Tp.Val460GluV460ENANA0.00130.000024750.015ARHL57Male2359891714:23598917 G / Ap.Thr402MetT402MNANA0.00470.000024710.015ARHL75Male2360864114:23608641 C / T (rs142951280)p.Val302IleV302I0.0005NA0.00470.00046130.015ARHL86Female2359887014:23598870 G / A (rs146946494) p.Arg418CysR418C0.0005NA0.0020.000024770.015control50Male2365210114:23652101 C / G (rs141772308)p.Arg8ProR8P0.0008NA0.00130.00081560.012control50Male2363562114:23635621 C / T (rs139927895)p.Ala94ThrA94T0.00120.0020.00130.002020.012control90Female2361236814:23612368 C / A (rs149245114)p.Arg185GlnR185LNANA0.0020.000024710.012 ARHL (age-related hearing loss). The age (years) of the subject when the Audiogram was performed is indicated. Variant [CHR: position reference/alternate (dbSNP135rsID)]. Consequence [HGUS annotation (protein change)]. Code [short description of the alternate variant]. Frequency of the mutations: Esp6500siv2 (NHLBI Exome Sequencing Project), 1000 g (1000 Genomes Project), Campion (The Allele Frequency Net Database) and ExAC (The Exome Aggregation Consortium). All the mutations found in SLC7A8 cases and controls from isolated villages of Friuli Venezia Giulia exhibited different frequencies in comparison to public data bases, such as ExAC among others (see Table 1). According to ExAC database’s constrain metrics (Lek et al., 2016), the gene shows evidence of tolerance of both loss of function (pLi = 0) and missense variation (missense Z score = −0.14). Functional studies of SLC7A8 mutations A structural model of human SLC7A8 protein built using the homologous protein AdiC (Kowalczyk et al., 2011) in the outward-facing conformation (Rosell et al., 2014) (Figure 5—figure supplement 1C and D) was used to localize all the mutations identified here. Interestingly, three of the four mutations found in ARHL patients were located in very striking places: (i) V302 is a conserved amino acid located in the extracellular loop four which corresponds to the external lid that closes the substrate binding site when the transporter is open to the cytosol, (ii) T402 is located in transmembrane (TM) domain 10 facing to the substrate binding site, and (iii) V460 is located at the very end of TM domain 12, with potential interaction with the plasma membrane. In contrast, R418 is in the intracellular loop 5, between TM domain 10 and TM domain 11 and with no functional role described in transporters with the LeuT-fold (Krishnamurthy and Gouaux, 2012). Thus, three of these mutations were promising candidates to affect the transporter function due to their crucial location. In vitro functional characterization of variants present in patients with ARHL and controls was performed by measuring amino acid uptake in HeLa cells co-transfected with the heavy subunit CD98hc and Strep tagged-SLC7A8 wild type and variants (Figure 5). Co-expression of the light (SLC7A8) and the heavy (CD98hc) subunits in the same cell increases the plasma membrane localization of the transporter (Rosell et al., 2014). All tested variants showed expression levels comparable to those of wild type, except for V460E that showed only 20% expression of wild -ype protein (Figure 5—source data 1), being the only variant that did not reach the plasma membrane as indicated by the lack of co-localization with wheat germ agglutinin staining (Figure 5A). Amino acid transport induced by SLC7A8 was analyzed for wild type and the identified variants (Figure 5B). All variants present in controls (R8P, R186L and A94T) conserved more than 80% of alanine transport compared with wild-type protein. Three variants found in patients with ARHL showed diminished alanine transport activity: T402M and V460E presented little residual transport activity (14.6 ± 2.6% and 3.6 ± 0.3% of wild-type activity, respectively) and R418C showed 50.7 ± 5.4% of wild-type alanine transport. Surprisingly, V302I presented similar alanine transport levels to wild type SLC7A8. Location of residue V302 within EL4 (within the external substrate lid (Figure 5—figure suppleme

Conference Article| May 01 1995 Intracellular mediators of insulin-like growth factor I during otic vesicle development YOLANDA LEON; YOLANDA LEON 1Instituto de Investigaciones Biomédicas, CSIC, Arturo Duperier 4, 28029 Madrid, SPAIN Search for other works by this author on: This Site PubMed Google Scholar CARMEN SANZ; CARMEN SANZ 1Instituto de Investigaciones Biomédicas, CSIC, Arturo Duperier 4, 28029 Madrid, SPAIN Search for other works by this author on: This Site PubMed Google Scholar ISABEL VARELA-NIETO ISABEL VARELA-NIETO 1Instituto de Investigaciones Biomédicas, CSIC, Arturo Duperier 4, 28029 Madrid, SPAIN Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1995) 23 (2): 185S. https://doi.org/10.1042/bst023185s Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation YOLANDA LEON, CARMEN SANZ, ISABEL VARELA-NIETO; Intracellular mediators of insulin-like growth factor I during otic vesicle development. Biochem Soc Trans 1 May 1995; 23 (2): 185S. doi: https://doi.org/10.1042/bst023185s Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1995 Biochemical Society1995 Article PDF first page preview Close Modal You do not currently have access to this content.

Introduction GPI Structure and Hydrolysis by Specific Phospholipases Diffusible Factors and the Regulation of GPI Levels IPG Structure and Biological Activities GPI/IPG Pathway and the Intracellular Signaling Circuit Acknowledgments References

Resumen del poster presentado al 3rd Symposium on Biomedical Research: Advances and Perspectives in Neuroscience, celebrado en la Universidad Autonoma de Madrid el 22 de abril de 2016.

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