Enhancement of the medial olivocochlear system prevents hidden hearing loss

Cochlear synaptopathy produced by exposure to noise levels that cause only transient auditory threshold elevations is a condition that affects many people and is believed to contribute to poor speech discrimination in noisy environments. These functional deficits in hearing, without changes in sensitivity, have been called hidden hearing loss (HHL). It has been proposed that activity of the medial olivocochlear (MOC) system can ameliorate acoustic trauma effects. Here we explore the role of the MOC system in HHL by comparing the performance of two different mouse models: an α9 nicotinic receptor subunit knock-out (KO; Chrna9 KO), which lacks cholinergic transmission between efferent neurons and hair cells; and a gain-of-function knock-in (KI; Chrna9L9′T KI) carrying an α9 point mutation that leads to enhanced cholinergic activity. Animals of either sex were exposed to sound pressure levels that in wild-type produced transient cochlear threshold shifts and a decrease in neural response amplitudes, together with the loss of ribbon synapses, which is indicative of cochlear synaptopathy. Moreover, a reduction in the number of efferent contacts to outer hair cells was observed. In Chrna9 KO ears, noise exposure produced permanent auditory threshold elevations together with cochlear synaptopathy. In contrast, the Chrna9L9′T KI was completely resistant to the same acoustic exposure protocol. These results show a positive correlation between the degree of HHL prevention and the level of cholinergic activity. Notably, enhancement of the MOC feedback promoted new afferent synapse formation, suggesting that it can trigger cellular and molecular mechanisms to protect and/or repair the inner ear sensory epithelium.Fil: Boero, Luis Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Castagna, Valeria Carolina. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Di Guilmi, Mariano Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Goutman, Juan Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Elgoyhen, Ana Belen. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Gomez Casati, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentin

Strengthening of the Efferent Olivocochlear System Leads to Synaptic Dysfunction and Tonotopy Disruption of a Central Auditory Nucleus

The auditory system in many mammals is immature at birth but precisely organized in adults. Spontaneous activity in the inner ear plays a critical role in guiding this maturation process. This is shaped by an efferent pathway that descends from the brainstem and makes transient direct synaptic contacts with inner hair cells. In this work, we used an α9 cholinergic nicotinic receptor knock-in mouse model (of either sex) with enhanced medial efferent activity (Chrna9L9'T, L9'T) to further understand the role of the olivocochlear system in the correct establishment of auditory circuits. Wave III of auditory brainstem responses (which represents synchronized activity of synapses within the superior olivary complex) was smaller in L9'T mice, suggesting a central dysfunction. The mechanism underlying this functional alteration was analyzed in brain slices containing the medial nucleus of the trapezoid body (MNTB), where neurons are topographically organized along a mediolateral (ML) axis. The topographic organization of MNTB physiological properties observed in wildtype (WT) was abolished in L9'T mice. Additionally, electrophysiological recordings in slices indicated MNTB synaptic alterations. In vivo multielectrode recordings showed that the overall level of MNTB activity was reduced in the L9'T The present results indicate that the transient cochlear efferent innervation to inner hair cells during the critical period before the onset of hearing is involved in the refinement of topographic maps as well as in setting the properties of synaptic transmission at a central auditory nucleus.SIGNIFICANCE STATEMENT Cochlear inner hair cells of altricial mammals display spontaneous electrical activity before hearing onset. The pattern and firing rate of these cells are crucial for the correct maturation of the central auditory pathway. A descending efferent innervation from the CNS contacts the hair cells during this developmental window. The present work shows that genetic enhancement of efferent function disrupts the orderly topographic distribution of biophysical and synaptic properties in the auditory brainstem and causes severe synaptic dysfunction. This work adds to the notion that the transient efferent innervation to the cochlea is necessary for the correct establishment of the central auditory circuitry.Fil: Di Guilmi, Mariano Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Boero, Luis Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia; ArgentinaFil: Castagna, Valeria C.. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia; ArgentinaFil: Rodríguez Contreras, Adrián. City University Of New York. The City College Of New York.; Estados UnidosFil: Wedemeyer, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Gomez Casati, Maria Eugenia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Elgoyhen, Ana Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia; Argentin

Inner Ear Genes Underwent Positive Selection and Adaptation in the Mammalian Lineage

The mammalian inner ear possesses functional and morphological innovations that contribute to its unique hearing capacities. The genetic bases underlying the evolution of this mammalian landmark are poorly understood. We propose that the emergence of morphological and functional innovations in the mammalian inner ear could have been driven by adaptive molecular evolution. In this work, we performed a meta-analysis of available inner ear gene expression data sets in order to identify genes that show signatures of adaptive evolution in the mammalian lineage. We analyzed ∼1,300 inner ear expressed genes and found that 13% show signatures of positive selection in the mammalian lineage. Several of these genes are known to play an important function in the inner ear. In addition, we identified that a significant proportion of genes showing signatures of adaptive evolution in mammals have not been previously reported to participate in inner ear development and/or physiology. We focused our analysis in two of these genes: STRIP2 and ABLIM2 by generating null mutant mice and analyzed their auditory function. We found that mice lacking Strip2 displayed a decrease in neural response amplitudes. In addition, we observed a reduction in the number of afferent synapses, suggesting a potential cochlear neuropathy. Thus, this study shows the usefulness of pursuing a high-throughput evolutionary approach followed by functional studies to track down genes that are important for inner ear function. Moreover, this approach sheds light on the genetic bases underlying the evolution of the mammalian inner ear.Fil: Pisciottano, Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Cinalli, Alejandro Raúl. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Stopiello, Juan Matías. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Castagna, Valeria Carolina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Elgoyhen, Ana Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Gomez Casati, Maria Eugenia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Franchini, Lucia Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

Preventing presbycusis in mice with enhanced medial olivocochlear feedback

"Growing old" is the most common cause of hearing loss. Agerelated hearing loss (ARHL) (presbycusis) first affects the ability to understand speech in background noise, even when auditory thresholds in quiet are normal. It has been suggested that cochlear denervation ("synaptopathy") is an early contributor to agerelated auditory decline. In the present work, we characterized age-related cochlear synaptic degeneration and hair cell loss in mice with enhanced α9α10 cholinergic nicotinic receptors gating kinetics ("gain of function" nAChRs). These mediate inhibitory olivocochlear feedback through the activation of associated calciumgated potassium channels. Cochlear function was assessed via distortion product otoacoustic emissions and auditory brainstem responses. Cochlear structure was characterized in immunolabeled organ of Corti whole mounts using confocal microscopy to quantify hair cells, auditory neurons, presynaptic ribbons, and postsynaptic glutamate receptors. Aged wild-type mice had elevated acoustic thresholds and synaptic loss. Afferent synapses were lost from inner hair cells throughout the aged cochlea, together with some loss of outer hair cells. In contrast, cochlear structure and function were preserved in aged mice with gain-of-function nAChRs that provide enhanced olivocochlear inhibition, suggesting that efferent feedback is important for long-term maintenance of inner ear function. Our work provides evidence that olivocochlear-mediated resistance to presbycusis-ARHL occurs via the α9α10 nAChR complexes on outer hair cells. Thus, enhancement of the medial olivocochlear system could be a viable strategy to prevent age-related hearing loss.Fil: Boero, Luis Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacología; ArgentinaFil: Castagna, Valeria Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacología; ArgentinaFil: Terreros, Gonzalo. Universidad de Chile; ChileFil: Moglie, Marcelo Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Silva, Sebastián. Universidad de Chile; ChileFil: Maass, Juan C.. Universidad de Chile; ChileFil: Fuchs, Paul A.. University Johns Hopkins; Estados UnidosFil: Delano, Paul H.. Universidad de Chile; ChileFil: Elgoyhen, Ana Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacología; ArgentinaFil: Gomez Casati, Maria Eugenia. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

GABA and ACh are co-released from olivocochlear efferent terminals

During development, inner hair cells (IHCs) in the mammalian cochlea are unresponsive to acoustic stimuli but instead present intrinsic electrical activity, crucial for the normal development of the auditory pathway. During this same period, neurons originating from the medial olivocochlear complex (MOC) transiently innervate IHCs. This innervation is mediated by acetylcholine (ACh), activating nicotinic receptors assembled by α9 and α10 subunits and is responsible for controlling IHC excitability during this period. Even though this is a cholinergic synapse, previous evidence indicates the presence of abundant GABA and GABAB receptors in MOC fibers in the inner spiral bundle. Moreover, the application of GABAB agonists reduces ACh release. Transgenic mice expressing channelrodhopsin (ChR2) under the control of either GAD or ChAT promoters were used in this study. Here we show for the first time, that optogenetically activated fibers in GAD-cre/ChR2 mice (n=7) produced postsynaptic responses that were blocked with cholinergic antagonists (n=3). In addition, pharmacological experiments indicate GABAB activation in ChAT-cre/ChR2 mice (n=4). ChAT-cre/TdTomato cochleas, co-stained with antibody against GAD, showed a co-localization of GABAergic and cholinergic terminals in the inner spiral bundle. Altogether these results strongly suggest that ACh is being co-released with GABA from MOC fibers.Fil: Castagnola, Tais. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Elgoyhen, Ana Belen. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Gomez Casati, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacología; ArgentinaFil: Castagna, Valeria Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacología; ArgentinaFil: Goutman, Juan Diego. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Wedemeyer, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaXXXVII Annual Meeting of the Argentine Society for Research in NeuroscienceCiudad Autónoma de Buenos AiresArgentinaSociedad Argentina de Investigación en Neurociencia

Synaptic activity at the MNTB is disrupted in a mouse model with enhanced efferent olivocochlear system

The auditory system in many mammals is immature at birth but precisely organized in adults. Spontaneous activity in the inner ear plays a critical role in guiding this process. This is shaped by an efferent pathway that descends from the brainstem and makes transient direct synaptic contacts with inner hair cells (IHCs). In this work, we used an β9 cholinergic receptor knock-in mouse model with enhanced medial efferent activity (Chrna9L9′T, L9′T) to understand the role of the olivocochlear system in the correct establishment of auditory circuits. Wave III amplitudes of auditory brainstem responses (which represent synchronized activity of synapses within the superior olivary complex) were smaller in L9′T mice, suggesting a central dysfunction. The mechanism underlying this functional alteration was analyzed in brain slices containing the medial nucleus of the trapezoid body (MNTB), where neurons are topographically organized along a medio-lateral axis. Electrophysiological recordings evidenced MNTB synaptic alterations. Spontaneous synaptic response (mEPSCs) displayed no changes in its amplitude among genotypes, while mEPSCs mean frequency displayed a significant increase in the L9’T lateral region (M: 2.52±0.56 Hz; L: 345 7.17±1.94 Hz; Mann-Whitney test, Z: -2.11, p=0.035). Moreover, evoked synaptic transmission was altered in the transgenic mice. While no significant differences in the unitary medial and lateral EPSC amplitudes were recorded in WT mice (M: 7.59±1.12 nA, n=9, 7 animals; L: 7.35±0.95 nA, n=10, 8 animals, ANOVA, F:0.027, p=0.87), evoked synaptic currents in the lateral side (5.07±0.87 nA, n=12, 11 animals) of L9’T mice were smaller compared to those of the medial side (8.05±1.37 nA, n=11, 11 animals; ANOVA, F:5.07, p=0.0357). These abnormalities were further supported by morphological alterations. Rhodamine-dextran labeling evidenced multiple innervation in L9’T MNTB principal cells suggesting an impairment during development. At the in-vivo level, multielectrode recordings showed that the overall level of MNTB activity was reduced in the L9’T. The average multi-unit activity in WT (11.49±3.58 Hz, n=6 animals) was larger than in L9’T mice (2.53-±0.43 Hz, n=8 animals; Mann-Whitney U Test, Z=2.19, 806 p=0.028). The present results suggest that the transient cochlear efferent innervation to IHCs during the critical period before the onset of hearing is involved in the refinement of topographic maps as well as in setting the correct synaptic transmission at central auditory nuclei.Fil: Di Guilmi, Mariano Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Boero, Luis Ezequiel. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Castagna, Valeria Carolina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Rodríguez Contreras, Adián. City University Of New York. The City College Of New York.; Estados UnidosFil: Wedemeyer, Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Gomez Casati, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia; ArgentinaFil: Elgoyhen, Ana Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaARO 43rd Annual MidWinter MeetingCaliforniaEstados UnidosAssociation for Research in Otolaryngolog

Accelerated Evolution Analysis Uncovers PKNOX2 as a Key Transcription Factor in the Mammalian Cochlea

The genetic bases underlying the evolution of morphological and functional innovations of the mammalian inner ear are poorly understood. Gene regulatory regions are thought to play an important role in the evolution of form and function. To uncover crucial hearing genes whose regulatory machinery evolved specifically in mammalian lineages, we mapped accelerated noncoding elements (ANCEs) in inner ear transcription factor (TF) genes and found that PKNOX2 harbors the largest number of ANCEs within its transcriptional unit. Using reporter gene expression assays in transgenic zebrafish, we determined that four PKNOX2-ANCEs drive differential expression patterns when compared with ortholog sequences from close outgroup species. Because the functional role of PKNOX2 in cochlear hair cells has not been previously investigated, we decided to study Pknox2 null mice generated by CRISPR/Cas9 technology. We found that Pknox2-/- mice exhibit reduced distortion product otoacoustic emissions (DPOAEs) and auditory brainstem response (ABR) thresholds at high frequencies together with an increase in peak 1 amplitude, consistent with a higher number of inner hair cells (IHCs)-auditory nerve synapsis observed at the cochlear basal region. A comparative cochlear transcriptomic analysis of Pknox2-/- and Pknox2+/+ mice revealed that key auditory genes are under Pknox2 control. Hence, we report that PKNOX2 plays a critical role in cochlear sensitivity at higher frequencies and that its transcriptional regulation underwent lineage-specific evolution in mammals. Our results provide novel insights about the contribution of PKNOX2 to normal auditory function and to the evolution of high-frequency hearing in mammals.Fil: Trigila, Anabella Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Castagna, Valeria Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia; ArgentinaFil: Berasain, Lara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Montini, Dante. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; ArgentinaFil: Gomez Casati, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia; ArgentinaFil: Franchini, Lucia Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentin

Cellular and functional mechanisms involved in hearing loss in DFNA2 mouse model

Function impairment in the voltage-gated K+ channel KCNQ4 is the main cause of DFNA2, a non-syndromic progressive hearing loss (HL). It occurs in two phases: initially, there is a mild HL at young ages, which then progresses to a profound HL in adulthood in the last phase. Previously, we reported that outer hair cell (OHC) death may contribute to the first phase and inner hair cell (IHC) and spiral ganglion neuron (SGN) degeneration would explain the last phase of DFNA2, in a mouse lacking KCNQ4 channel (Kcnq4-/- ). Now we correlate these findings with the molecular and functional alterations in this mouse model of HL. In 3-6 weeks-old (W) Kcnq4-/- animals, using immunofluorescence (IF), we found an increase of cleaved caspase-3 (CAS-3) expression in the OHCs area in the basal turn. Moreover, gene expression analysis by qPCR in young Kcnq4-/- mice revealed that pro-apoptotic Bax transcript level was ~6-fold higher than in the WT animals, while anti-apoptotic Bcl2 gene expression was drastically reduced. Additionally, by IF, we found a lower synaptic density and mislocalization of the efferent terminals that contact OHCs from Kcnq4-/- mice. Previous studies showed that this model has an increase in the hearing threshold at low frequencies but with no decrease in IHC number. However, using the C3H mouse strain, we found loss of IHCs and SGNs in 1-year-old mice lacking KCNQ4 expression. To assess the auditory function in middle-aged mice, we initially performed the Preyer’s reflex test. We determined that ~50% of Kcnq4-/- mice did not pass the test, indicating a profound HL. Auditory brainstem response (ABR) test exhibited a significant auditory threshold shift of ~60 dB SPL in the 5.6-45.25 kHz frequency range, pointing out that the electric transmission through the whole auditory pathway is affected by KCNQ4 absence. Following this, we observed CAS-3 expression in SGNs at 1-year-old mice. IHCs neither express CAS-3 nor the autophagy marker LC3-B2. However, they showed by scanning electron microscopy (SEM), different stereocilia alterations like fusion and missing ones in middle-aged Kcnq4-/- mice. Distortion product of otoacoustic emissions (DPOAE) test revealed an auditory threshold shift of ~20-30 dB SPL in the 8-32 kHz range, indicating that OHCs function is severely impaired in these mice. Despite this, cochlear microphonic signals were detected mainly at low frequencies, suggesting a mild activity of OHCs in the apical turn. Our results demonstrated that during the first stage of DFNA2, OHCs die by apoptosis while efferent synapsis is disorganized. In the second phase, apoptosis is present in SGNs but not in IHCs which are also lost. However, we found diverse stereocilia defects, which could account for their lack of auditory signal generation in middle-aged Kcnq4-/- mice. Collectively, these findings may help to understand the cellular and molecular mechanisms underlying the biphasic HL.Fil: Rias, Ezequiel Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Carignano, Camila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Dionisio, Leonardo Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Castagna, Valeria Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Stupniki Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Gomez Casati, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Spitzmaul, Guillermo Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaLVII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research and XVI Annual Meeting of the Argentinean Society for General MicrobiologyBuenos AiresArgentinaSociedad Argentina de Investigación Bioquímica y Biología MolecularSociedad Argentina de Microbiología Genera

KCNQ4 channel is required for outer hair cell survival, postnatal maturation and efferent innervation

INTRODUÇÃO: KCNQ4 is a voltage-gated K+ channel essential for hearing. Impairment of its function produces a chronic depolarization of hair cells (HC) leading to cell death, and hearing loss (HL). The mechanism of cell death remains unknown. A protective pathway is carried out by the efferent system by the activation of its synapses, restoring membrane potential. However, its contribution to KCNQ4-related HL is unknown. OBJETIVOS: Our aim is to evaluate the molecular, tissue and functional alterations of HCs and their efferent connectivity in a mouse model of HL, which is a knock-out for the KCNQ4 channel (Kcnq4-/- ) (Nº083/2016). MÉTODOS: In the WT and Kcnq4-/- mice we performed immunofluorescence (IF) combined with superresolution-confocal microscopy, quantitative PCR (qPCR), Auditory brainstem response (ABR) and Distortion product of otoacoustic emissions (DPOAE) assays. RESULTADOS: In 4 postnatal-weeks-old (W) Kcnq4-/- animals, using IF, we found an increase of cleaved CASPASE-3 expression in outer hair cells (OHC) from the basal turn. Moreover, qPCR analysis revealed that the expression ratio between the pro- and anti-apoptotic factors Bax/Bcl2 was ~76-fold higher. We also found mislocalization of the membrane protein PRESTIN and of the efferent synapses (~30%) that contact OHC. Furthermore, we found no changes in terminals volume but a 40% decrease in the number of synapses/OHC in Kcnq4-/- mice. By contrast, both genotypes at 2W exhibited the same immature wiring pattern. To test the hearing function, we performed ABR showing a significant threshold shift of 20-48 dB SPL in the 5.6-45.25 kHz frequency range. Besides, DPOAE test revealed a threshold shift of ~12-20 dB SPL in the 8-45.25 kHz range. CONCLUSÕES: We found new insights into the mechanism of HL in the Kcnq4-/- mice. While the basal OHCs die by apoptosis, hearing function is impaired in all cochlear regionsFil: Spitzmaul, Guillermo Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Rias, Ezequiel Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Carignano, Camila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Castagna, Valeria Carolina. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Vera, Marcela Sonia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Stupniki, Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; ArgentinaFil: Gomez Casati, Maria Eugenia. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Dionisio, Leonardo Raul. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia; Argentina3rd. Federación de Asociaciones Latinoamericanas y del Caribe de Neurociencias CongressBelémBrasilFederación de Asociaciones Latinoamericanas y del Caribe de Neurociencia