Phytosterols reverse antiretroviral-induced hearing loss, with potential implications for cochlear aging.

Cholesterol contributes to neuronal membrane integrity, supports membrane protein clustering and function, and facilitates proper signal transduction. Extensive evidence has shown that cholesterol imbalances in the central nervous system occur in aging and in the development of neurodegenerative diseases. In this work, we characterize cholesterol homeostasis in the inner ear of young and aged mice as a new unexplored possibility for the prevention and treatment of hearing loss. Our results show that cholesterol levels in the inner ear are reduced during aging, an effect that is associated with an increased expression of the cholesterol 24-hydroxylase (CYP46A1), the main enzyme responsible for cholesterol turnover in the brain. In addition, we show that pharmacological activation of CYP46A1 with the antiretroviral drug efavirenz reduces the cholesterol content in outer hair cells (OHCs), leading to a decrease in prestin immunolabeling and resulting in an increase in the distortion product otoacoustic emissions (DPOAEs) thresholds. Moreover, dietary supplementation with phytosterols, plant sterols with structure and function similar to cholesterol, was able to rescue the effect of efavirenz administration on the auditory function. Altogether, our findings point towards the importance of cholesterol homeostasis in the inner ear as an innovative therapeutic strategy in preventing and/or delaying hearing loss.

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.

Preventing presbycusis in mice with enhanced medial olivocochlear feedback.

"Growing old" is the most common cause of hearing loss. Age-related 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 age-related 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 calcium-gated 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.

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.

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.

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.SIGNIFICANCE STATEMENT Noise overexposure is a major cause of a variety of perceptual disabilities, including speech-in-noise difficulties, tinnitus, and hyperacusis. Here we show that exposure to noise levels that do not cause permanent threshold elevations or hair cell death can produce a loss of cochlear nerve synapses to inner hair cells as well as degeneration of medial olivocochlear (MOC) terminals contacting the outer hair cells. Enhancement of the MOC reflex can prevent both types of neuropathy, highlighting the potential use of drugs that increase α9α10 nicotinic cholinergic receptor activity as a pharmacotherapeutic strategy to avoid hidden hearing loss.

Activation of presynaptic GABA(B(1a,2)) receptors inhibits synaptic transmission at mammalian inhibitory cholinergic olivocochlear-hair cell synapses.

The synapse between olivocochlear (OC) neurons and cochlear mechanosensory hair cells is cholinergic, fast, and inhibitory. The inhibitory sign of this cholinergic synapse is accounted for by the activation of Ca(2+)-permeable postsynaptic α9α10 nicotinic receptors coupled to the opening of hyperpolarizing Ca(2+)-activated small-conductance type 2 (SK2)K(+) channels. Acetylcholine (ACh) release at this synapse is supported by both P/Q- and N-type voltage-gated calcium channels (VGCCs). Although the OC synapse is cholinergic, an abundant OC GABA innervation is present along the mammalian cochlea. The role of this neurotransmitter at the OC efferent innervation, however, is for the most part unknown. We show that GABA fails to evoke fast postsynaptic inhibitory currents in apical developing inner and outer hair cells. However, electrical stimulation of OC efferent fibers activates presynaptic GABA(B(1a,2)) receptors [GABA(B(1a,2))Rs] that downregulate the amount of ACh released at the OC-hair cell synapse, by inhibiting P/Q-type VGCCs. We confirmed the expression of GABA(B)Rs at OC terminals contacting the hair cells by coimmunostaining for GFP and synaptophysin in transgenic mice expressing GABA(B1)-GFP fusion proteins. Moreover, coimmunostaining with antibodies against the GABA synthetic enzyme glutamic acid decarboxylase and synaptophysin support the idea that GABA is directly synthesized at OC terminals contacting the hair cells during development. Thus, we demonstrate for the first time a physiological role for GABA in cochlear synaptic function. In addition, our data suggest that the GABA(B1a) isoform selectively inhibits release at efferent cholinergic synapses.

Noise Exposure Potentiates Exocytosis From Cochlear Inner Hair Cells.

Noise-induced hearing loss has gained relevance as one of the most common forms of hearing impairment. The anatomical correlates of hearing loss, principally cell damage and/or death, are relatively well-understood histologically. However, much less is known about the physiological aspects of damaged, surviving cells. Here we addressed the functional consequences of noise exposure on the capacity of inner hair cells (IHCs) to release synaptic vesicles at synapses with spiral ganglion neurons (SGNs). Mice of either sex at postnatal day (P) 15-16 were exposed to 1-12 kHz noise at 120 dB sound pressure level (SPL), for 1 h. Exocytosis was measured by tracking changes in membrane capacitance (ΔCm) from IHCs of the apical cochlea. Upon IHC depolarization to different membrane potentials, ΔC m showed the typical bell-shaped curve that mirrors the voltage dependence of Ca2+ influx, in both exposed and unexposed cells. Surprisingly, from IHCs at 1-day after exposure (d.a.e.), we found potentiation of exocytosis at the peak of the bell-shaped curve. The increase in exocytosis was not accompanied by changes in whole-cell Ca2+ influx, suggesting a modification in coupling between Ca2+ channels and synaptic vesicles. Consistent with this notion, noise exposure also changed the Ca2+-dependence of exocytosis from linear to supralinear. Noise exposure did not cause loss of IHCs, but did result in a small reduction in the number of IHC-SGN synapses at 1-d.a.e. which recovered by 14-d.a.e. In contrast, a strong reduction in auditory brainstem response wave-I amplitude (representing synchronous firing of SGNs) and distortion product otoacoustic emissions (reflecting outer hair cell function) indicated a profound hearing loss at 1- and 14-d.a.e. To determine the role of glutamate release in the noise-induced potentiation of exocytosis, we evaluated vesicular glutamate transporter-3 (Vglut3) knock-out (KO) mice. Unlike WT, IHCs from Vglut3 KO mice showed a noise-induced reduction in ΔC m and Ca2+ influx with no change in the Ca2+-dependence of exocytosis. Together, these results indicate that traumatic noise exposure triggers changes of IHC synaptic function including a Vglut3-dependent potentiation of exocytosis.

Exposure of Developing Male Rats to One or Multiple Noise Sessions and Different Housing Conditions: Hippocampal Thioredoxin Changes and Behavioral Alterations.

Exposure of developing rats to noise has shown to induce hippocampal-related behavioral alterations that were prevented after a week of housing in an enriched environment. However, neither the effect of repeated exposures nor its impact on key endogenous antioxidants had been studied yet. Thus, the aim of the present work was to reveal novel data about hippocampal oxidative state through the measurement of possible age-related differences in the levels of hippocampal thioredoxins in rats exposed to noise at different developmental ages and subjected to different schemes and housing conditions. In addition, the possibility that oxidative changes could underlie hippocampal-related behavioral changes was also analyzed. Developing male Wistar rats were exposed to noise for 2 h, either once or for 5 days. Upon weaning, some animals were transferred to an enriched cage for 1 week, whereas others were kept in standard cages. One week later, auditory and behavioral assessments, as well as measurement of hippocampal thioredoxin, were performed. Whereas no changes in the auditory function were observed, significant behavioral differences were found, that varied according to the age, scheme of exposure and housing condition. In addition, a significant increase in Trx-1 levels was found in all noise-exposed groups housed in standard cages. Housing animals in an enriched environment for 1 week was effective in preventing most of these changes. These findings suggest that animals become less susceptible to undergo behavioral alterations after repeated exposure to an environmental challenge, probably due to the ability of adaptation to an unfavorable condition. Moreover, it could be hypothesized that damage to younger individuals could be more easily prevented by a housing manipulation.

Cochlear hair cells: The sound-sensing machines.

The sensory epithelium of the mammalian inner ear contains two types of mechanosensory cells: inner (IHC) and outer hair cells (OHC). They both transduce mechanical force generated by sound waves into electrical signals. In their apical end, these cells possess a set of stereocilia representing the mechanosensing organelles. IHC are responsible for detecting sounds and transmitting the acoustic information to the brain by converting graded depolarization into trains of action potentials in auditory nerve fibers. OHC are responsible for the active mechanical amplification process that leads to the fine tuning and high sensitivity of the mammalian inner ear. This active amplification is the consequence of the ability of OHC to alter their cell length in response to changes in membrane potential, and is controlled by an efferent inhibitory innervation. Medial olivocochlear efferent fibers, originating in the brainstem, synapse directly at the base of OHC and release acetylcholine. A very special type of nicotinic receptor, assembled by α9α10 subunits, participates in this synapse. Here we review recent knowledge and the role of both afferent and efferent synapse in the inner ear.

Cell-specific inducible gene recombination in postnatal inner ear supporting cells and glia.

Recent studies indicate that supporting cells play important roles in inner ear development, function, and regeneration after injury, but the molecular mechanisms underlying these processes remain poorly understood. Inducible cell-specific gene recombination in supporting cells could be a powerful tool to study the roles of specific molecules in these cells. Here we tested the feasibility, effectiveness, and cell specificity of inducible Cre-mediated gene recombination in the postnatal inner ear using mice that express an inducible form of Cre (CreER(T)) under the transcriptional control of the proteolipid protein (PLP) promoter. We assessed the pattern of tamoxifen-induced gene recombination in the inner ear using the ROSA26-LacZ reporter line, in which the beta-galactosidase gene is expressed only after Cre-mediated excision of a loxP-flanked stop cassette. Recombination was detected in cochlear inner phalangeal cells, supporting cells surrounding hair cells in vestibular maculae and cristae. Recombination also occurred in Schwann cells. We also found that this CreER(T) line can be used to increase and decrease the levels of expression of a trophic factor, brain-derived neurotrophic factor, specifically in supporting cells. These results show that PLP/CreER(T) mice are a powerful tool to dissect gene function in inner ear supporting cells.

Electrical properties and functional expression of ionic channels in cochlear inner hair cells of mice lacking the alpha10 nicotinic cholinergic receptor subunit.

Cochlear inner hair cells (IHCs) release neurotransmitter onto afferent auditory nerve fibers in response to sound stimulation. During early development, synaptic transmission is triggered by spontaneous Ca2+ spikes which are modulated by an efferent cholinergic innervation to IHCs. This synapse is inhibitory and mediated by the alpha9alpha10 nicotinic cholinergic receptor (nAChR). After the onset of hearing, large-conductance Ca2+-activated K+ channels are acquired and both the spiking activity and the efferent innervation disappear from IHCs. In this work, we studied the developmental changes in the membrane properties of cochlear IHCs from alpha10 nAChR gene (Chrna10) "knockout" mice. Electrophysiological properties of IHCs were studied by whole-cell recordings in acutely excised apical turns of the organ of Corti from developing mice. Neither the spiking activity nor the developmental functional expression of voltage-gated and/or calcium-sensitive K+ channels is altered in the absence of the alpha10 nAChR subunit. The present results show that the alpha10 nAChR subunit is not essential for the correct establishment of the intrinsic electrical properties of IHCs during development.

Alpha-RgIA: a novel conotoxin that specifically and potently blocks the alpha9alpha10 nAChR.

The alpha9 and alpha10 nicotinic acetylcholine receptor (nAChR) subunits assemble to form the alpha9alpha10 nAChR subtype. This receptor is believed to mediate cholinergic synaptic transmission between efferent olivocochlear fibers and the hair cells of the cochlea. In addition alpha9 and/or alpha10 expression has been described in dorsal root ganglion neurons, lymphocytes, skin keratinocytes, and the pars tuberalis of the pituitary. Specific antagonists that selectively block the alpha9alpha10 channel could be valuable tools for elucidating its role in these diverse tissues. This study describes a novel alpha-conotoxin from the Western Atlantic species Conus regius, alpha-conotoxin RgIA (alpha-RgIA), that is a subtype specific blocker of the alpha9alpha10 nAChR. alpha-RgIA belongs to the alpha4/3 subfamily of the alpha-conotoxin family; sequence and subtype specificity comparisons between alpha-RgIA and previously characterized alpha4/3 toxins indicate that the amino acids in the C-terminal half of alpha-RgIA are responsible for its preferential inhibition of the alpha9alpha10 nAChR subtype.

Biophysical and pharmacological characterization of nicotinic cholinergic receptors in rat cochlear inner hair cells.

Before the onset of hearing, a transient efferent innervation is found on inner hair cells (IHCs). This synapse is inhibitory and mediated by a nicotinic cholinergic receptor (nAChR) probably formed by the alpha9 and alpha10 subunits. We analysed the pharmacological and biophysical characteristics of the native nAChR using whole-cell recordings from IHCs in acutely excised apical turns of the rat organ of Corti. Nicotine did not activate but rather blocked the acetylcholine (ACh)-evoked currents with an IC50 of 1 +/- 0.1 microM. Antagonists of non-cholinergic receptors such as strychnine, bicuculline and ICS-205930 blocked ACh-evoked responses with an IC50 of 8.6 +/- 0.8 nM, 59 +/- 4 nM and 0.30 +/- 0.02 microM, respectively. The IHC nAChR was both permeable to (P(Ca)/P(Na) = 8 +/- 0.9) and modulated by external Ca2+. ACh-evoked currents were potentiated by Ca2+ up to 500 microM but were reduced by higher concentrations of this cation. Ba2+ mimicked the effects of Ca2+ whereas Mg2+ only blocked these currents. In addition, elevation of extracellular Ca2+ reduced the amplitude of spontaneous synaptic currents without affecting their time course. The receptor had an EC50 for ACh of 60.7 +/- 2.8 microM in 0.5 mM Ca2+. In the absence of Ca2+, the EC50 for ACh increased, suggesting that potentiation by Ca2+ involves changes in the apparent affinity for the agonist. These pharmacological and biophysical characteristics of the IHC nAChR closely resemble those of the recombinant alpha9alpha10 nAChR, reinforcing the hypothesis that the functional nAChR at the olivocochlear efferent-IHC synapse is composed of both the alpha9 and alpha10 subunits.