Thyroid hormone controls the timing of cochlear ribbon synapse maturation.

Ribbon synapses in the cochlear hair cells are subject to extensive pruning and maturation processes before hearing onset. Previous studies have highlighted the pivotal role of thyroid hormone (TH) in this developmental process, yet the detailed mechanisms are largely unknown. In this study, we found that the thyroid hormone receptor α (Thrα) is expressed in both sensory epithelium and spiral ganglion neurons in mice. Hypothyroidism, induced by Pax8 gene knockout, significantly delays the synaptic pruning during postnatal development in mice. Detailed spatiotemporal analysis of ribbon synapse distribution reveals that synaptic maturation involves not only ribbon pruning but also their migration, both of which are notably delayed in the cochlea of Pax8 knockout mice. Intriguingly, postnatal hyperthyroidism, induced by intraperitoneal injections of liothyronine sodium (T3), accelerates the pruning of ribbon synapses to the mature state without affecting the auditory functions. Our findings suggest that thyroid hormone does not play a deterministic role but rather controls the timing of cochlear ribbon synapse maturation.

TMEM63B channel is the osmosensor required for thirst drive of interoceptive neurons.

Thirst plays a vital role in the regulation of body fluid homeostasis and if deregulated can be life-threatening. Interoceptive neurons in the subfornical organ (SFO) are intrinsically osmosensitive and their activation by hyperosmolarity is necessary and sufficient for generating thirst. However, the primary molecules sensing systemic osmolarity in these neurons remain elusive. Here we show that the mechanosensitive TMEM63B cation channel is the osmosensor required for the interoceptive neurons to drive thirst. TMEM63B channel is highly expressed in the excitatory SFO thirst neurons. TMEM63B deletion in these neurons impaired hyperosmolarity-induced drinking behavior, while re-expressing TMEM63B in SFO restored water appetite in TMEM63B-deficient mice. Remarkably, hyperosmolarity activates TMEM63B channels, leading to depolarization and increased firing rate of the interoceptive neurons, which drives drinking behavior. Furthermore, TMEM63B deletion did not affect sensitivities of the SFO neurons to angiotensin II or hypoosmolarity, suggesting that TMEM63B plays a specialized role in detecting hyperosmolarity in SFO neurons. Thus, our results reveal a critical osmosensor molecule for the generation of thirst perception.

Cingulin regulates hair cell cuticular plate morphology and is required for hearing in human and mouse.

Cingulin (CGN) is a cytoskeleton-associated protein localized at the apical junctions of epithelial cells. CGN interacts with major cytoskeletal filaments and regulates RhoA activity. However, physiological roles of CGN in development and human diseases are currently unknown. Here, we report a multi-generation family presenting with autosomal dominant non-syndromic hearing loss (ADNSHL) that co-segregates with a CGN heterozygous truncating variant, c.3330delG (p.Leu1110Leufs*17). CGN is normally expressed at the apical cell junctions of the organ of Corti, with enriched localization at hair cell cuticular plates and circumferential belts. In mice, the putative disease-causing mutation results in reduced expression and abnormal subcellular localization of the CGN protein, abolishes its actin polymerization activity, and impairs the normal morphology of hair cell cuticular plates and hair bundles. Hair cell-specific Cgn knockout leads to high-frequency hearing loss. Importantly, Cgn mutation knockin mice display noise-sensitive, progressive hearing loss and outer hair cell degeneration. In summary, we identify CGN c.3330delG as a pathogenic variant for ADNSHL and reveal essential roles of CGN in the maintenance of cochlear hair cell structures and auditory function.

A splicing-dependent ER retention signal regulates surface expression of the mechanosensitive TMEM63B cation channel.

TMEM63B is a mechanosensitive cation channel activated by hypoosmotic stress and mechanic stimulation. We recently reported a brain-specific alternative splicing of exon 4 in TMEM63B. The short variant lacking exon 4, which constitutes the major isoform in the brain, exhibits enhanced responses to hypoosmotic stimulation compared to the long isoform containing exon 4. However, the mechanisms affecting this differential response are unclear. Here, we showed that the short isoform exhibited stronger cell surface expression compared to the long variant. Using mutagenesis screening of the coding sequence of exon 4, we identified an RXR-type endoplasmic reticulum (ER) retention signal (RER). We found that this motif was responsible for binding to the COPI retrieval vesicles, such that the longer TMEM63B isoforms were more likely to be retrotranslocated to the ER than the short isoforms. In addition, we demonstrated long TMEM63Bs could form heterodimers with short isoforms and reduce their surface expression. Taken together, our findings revealed an ER retention signal in the alternative splicing domain of TMEM63B that regulates the surface expression of TMEM63B protein and channel function.

Heterogeneity analysis of astrocytes following spinal cord injury at single-cell resolution.

Astrocytes play many important functions in response to spinal cord injury (SCI) in an activated manner, including clearance of necrotic tissue, formation of protective barrier, maintenance of microenvironment balance, interaction with immune cells, and formation of the glial scar. More and more studies have shown that the astrocytes are heterogeneous, such as inflammatory astrocyte 1 (A1) and neuroprotective astrocyte 2 (A2) types. However, the subtypes of astrocyte resulting from SCI have not been clearly defined. In this study, using single-cell RNA sequencing, we constructed the transcriptomic profile of astrocytes from uninjured spinal cord tissue and injured tissue nearby the lesion epicenter at 0.5, 1, 3, 7, 14, 60, and 90 days after mouse hemisection spinal cord surgery. Our analysis uncovered six transcriptionally distinct astrocyte states, including Atp1b2+ , S100a4+ , Gpr84+ , C3+ /G0s2+ , GFAP+ /Tm4sf1+ , and Gss+ /Cryab+ astrocytes. We used these new signatures combined with canonical astrocyte markers to determine the distribution of morphologically and physiologically distinct astrocyte population at injured sites by immunofluorescence staining. Then we identified the dynamic evolution process of each astrocyte subtype following SCI. Finally, we also revealed the evolution of highly expressed genes in these astrocyte subtypes at different phases of SCI. Together, we provided six astrocyte subtypes at single-cell resolution following SCI. These data not only contribute to understand the heterogeneity of astrocytes during SCI but also help to find new astrocyte subtypes as a target for SCI repair.

Macrophages Are Dispensable for Postnatal Pruning of the Cochlear Ribbon Synapses.

Ribbon synapses of cochlear hair cells undergo pruning and maturation before the hearing onset. In the central nervous system (CNS), synaptic pruning was mediated by microglia, the brain-resident macrophages, via activation of the complement system. Whether a similar mechanism regulates ribbon synapse pruning is currently unknown. In this study, we report that the densities of cochlear macrophages surrounding hair cells were highest at around P8, corresponding well to the completion of ribbon synaptic pruning by P8-P9. Surprisingly, using multiple genetic mouse models, we found that postnatal pruning of the ribbon synapses and auditory functions were unaffected by the knockout of the complement receptor 3 (CR3) or by ablations of macrophages expressing either LysM or Cx3cr1. Our results suggest that unlike microglia in the CNS, macrophages in the cochlea do not mediate pruning of the cochlear ribbon synapses.

Cochlear Sox2+ Glial Cells Are Potent Progenitors for Spiral Ganglion Neuron Reprogramming Induced by Small Molecules.

In the mammalian cochlea, spiral ganglion neurons (SGNs) relay the acoustic information to the central auditory circuits. Degeneration of SGNs is a major cause of sensorineural hearing loss and severely affects the effectiveness of cochlear implant therapy. Cochlear glial cells are able to form spheres and differentiate into neurons in vitro. However, the identity of these progenitor cells is elusive, and it is unclear how to differentiate these cells toward functional SGNs. In this study, we found that Sox2+ subpopulation of cochlear glial cells preserves high potency of neuronal differentiation. Interestingly, Sox2 expression was downregulated during neuronal differentiation and Sox2 overexpression paradoxically inhibited neuronal differentiation. Our data suggest that Sox2+ glial cells are potent SGN progenitor cells, a phenotype independent of Sox2 expression. Furthermore, we identified a combination of small molecules that not only promoted neuronal differentiation of Sox2- glial cells, but also removed glial cell identity and promoted the maturation of the induced neurons (iNs) toward SGN fate. In summary, we identified Sox2+ glial subpopulation with high neuronal potency and small molecules inducing neuronal differentiation toward SGNs.

High-throughput screening on cochlear organoids identifies VEGFR-MEK-TGFB1 signaling promoting hair cell reprogramming.

Hair cell degeneration is a major cause of sensorineural hearing loss. Hair cells in mammalian cochlea do not spontaneously regenerate, posing a great challenge for restoration of hearing. Here, we establish a robust, high-throughput cochlear organoid platform that facilitates 3D expansion of cochlear progenitor cells and differentiation of hair cells in a temporally regulated manner. High-throughput screening of the FDA-approved drug library identified regorafenib, a VEGFR inhibitor, as a potent small molecule for hair cell differentiation. Regorafenib also promotes reprogramming and maturation of hair cells in both normal and neomycin-damaged cochlear explants. Mechanistically, inhibition of VEGFR suppresses TGFB1 expression via the MEK pathway and TGFB1 downregulation directly mediates the effect of regorafenib on hair cell reprogramming. Our study not only demonstrates the power of a cochlear organoid platform in high-throughput analyses of hair cell physiology but also highlights VEGFR-MEK-TGFB1 signaling crosstalk as a potential target for hair cell regeneration and hearing restoration.

Aldh inhibitor restores auditory function in a mouse model of human deafness.

Genetic hearing loss is a common health problem with no effective therapy currently available. DFNA15, caused by mutations of the transcription factor POU4F3, is one of the most common forms of autosomal dominant non-syndromic deafness. In this study, we established a novel mouse model of the human DFNA15 deafness, with a Pou4f3 gene mutation (Pou4f3Δ) identical to that found in a familial case of DFNA15. The Pou4f3(Δ/+) mice suffered progressive deafness in a similar manner to the DFNA15 patients. Hair cells in the Pou4f3(Δ/+) cochlea displayed significant stereociliary and mitochondrial pathologies, with apparent loss of outer hair cells. Progression of hearing and outer hair cell loss of the Pou4f3(Δ/+) mice was significantly modified by other genetic and environmental factors. Using Pou4f3(-/+) heterozygous knockout mice, we also showed that DFNA15 is likely caused by haploinsufficiency of the Pou4f3 gene. Importantly, inhibition of retinoic acid signaling by the aldehyde dehydrogenase (Aldh) and retinoic acid receptor inhibitors promoted Pou4f3 expression in the cochlear tissue and suppressed the progression of hearing loss in the mutant mice. These data demonstrate Pou4f3 haploinsufficiency as the main underlying cause of human DFNA15 deafness and highlight the therapeutic potential of Aldh inhibitors for treatment of progressive hearing loss.

The Cation Channel TMEM63B Is an Osmosensor Required for Hearing.

Hypotonic stress causes the activation of swelling-activated nonselective cation channels (NSCCs), which leads to Ca2+-dependent regulatory volume decrease (RVD) and adaptive maintenance of the cell volume; however, the molecular identities of the osmosensitive NSCCs remain unclear. Here, we identified TMEM63B as an osmosensitive NSCC activated by hypotonic stress. TMEM63B is enriched in the inner ear sensory hair cells. Genetic deletion of TMEM63B results in necroptosis of outer hair cells (OHCs) and progressive hearing loss. Mechanistically, the TMEM63B channel mediates hypo-osmolarity-induced Ca2+ influx, which activates Ca2+-dependent K+ channels required for the maintenance of OHC morphology. These findings demonstrate that TMEM63B is an osmosensor of the mammalian inner ear and the long-sought cation channel mediating Ca2+-dependent RVD.

Hidden Hearing Loss: A Disorder with Multiple Etiologies and Mechanisms.

Hidden hearing loss (HHL), a recently described auditory disorder, has been proposed to affect auditory neural processing and hearing acuity in subjects with normal audiometric thresholds, particularly in noisy environments. In contrast to central auditory processing disorders, HHL is caused by defects in the cochlea, the peripheral auditory organ. Noise exposure, aging, ototoxic drugs, and peripheral neuropathies are some of the known risk factors for HHL. Our knowledge of the causes and mechanisms of HHL are based primarily on animal models. However, recent clinical studies have also shed light on the etiology and prevalence of this cochlear disorder and how it may affect auditory perception in humans. Here, we review the current knowledge regarding the causes and cellular mechanisms of HHL, summarize information on available noninvasive tests for differential diagnosis, and discuss potential therapeutic approaches for treatment of HHL.

Synaptopathy as a Mechanism for Age-Related Vestibular Dysfunction in Mice.

Age-related decline of inner ear function contributes to both hearing loss and balance disorders, which lead to impaired quality of life and falls that can result in injury and even death. The cellular mechanisms responsible for the ear's functional decline have been controversial, but hair cell loss has been considered the key cause for a long time. However, recent studies showed that in the cochlea, loss of inner hair cell (IHC) synapses precedes hair cell or neuronal loss, and this synaptopathy is an early step in the functional decline. Whether a similar process occurs in the vestibular organ, its timing and its relationship to organ dysfunction remained unknown. We compared the time course of age-related deterioration in vestibular and cochlear functions in mice as well as characterized the age-associated changes in their utricles at the histological level. We found that in the mouse, as in humans, age-related decline in vestibular evoked potentials (VsEPs) occurs later than hearing loss. As in the cochlea, deterioration of VsEPs correlates with the loss of utricular ribbon synapses but not hair cells or neuronal cell bodies. Furthermore, the age-related synaptic loss is restricted to calyceal innervations in the utricular extrastriolar region. Hence, our findings suggest that loss of extrastriolar calyceal synapses has a key role in age-related vestibular dysfunction (ARVD).

Auditory metabolomics, an approach to identify acute molecular effects of noise trauma.

Animal-based studies have provided important insights into the structural and functional consequences of noise exposure on the cochlea. Yet, less is known about the molecular mechanisms by which noise induces cochlear damage, particularly at relatively low exposure levels. While there is ample evidence that noise exposure leads to changes in inner ear metabolism, the specific effects of noise exposure on the cochlear metabolome are poorly understood. In this study we applied liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS)-based metabolomics to analyze the effects of noise on the mouse inner ear. Mice were exposed to noise that induces temporary threshold shifts, synaptopathy and permanent hidden hearing loss. Inner ears were harvested immediately after exposure and analyzed by targeted metabolomics for the relative abundance of 220 metabolites across the major metabolic pathways in central carbon metabolism. We identified 40 metabolites differentially affected by noise. Our approach detected novel noise-modulated metabolites and pathways, as well as some already linked to noise exposure or cochlear function such as neurotransmission and oxidative stress. Furthermore, it showed that metabolic effects of noise on the inner ear depend on the intensity and duration of exposure. Collectively, our results illustrate that metabolomics provides a powerful approach for the characterization of inner ear metabolites affected by auditory trauma. This type of information could lead to the identification of drug targets and novel therapies for noise-induced hearing loss.

PlexinA2 Forward Signaling through Rap1 GTPases Regulates Dentate Gyrus Development and Schizophrenia-like Behaviors.

Dentate gyrus (DG) development requires specification of granule cell (GC) progenitors in the hippocampal neuroepithelium, as well as their proliferation and migration into the primordial DG. We identify the Plexin family members Plxna2 and Plxna4 as important regulators of DG development. Distribution of immature GCs is regulated by Sema5A signaling through PlxnA2 and requires a functional PlxnA2 GTPase-activating protein (GAP) domain and Rap1 small GTPases. In adult Plxna2-/- but not Plxna2-GAP-deficient mice, the dentate GC layer is severely malformed, neurogenesis is compromised, and mossy fibers form aberrant synaptic boutons within CA3. Behavioral studies with Plxna2-/- mice revealed deficits in associative learning, sociability, and sensorimotor gating-traits commonly observed in neuropsychiatric disorder. Remarkably, while morphological defects are minimal in Plxna2-GAP-deficient brains, defects in fear memory and sensorimotor gating persist. Since allelic variants of human PLXNA2 and RAP1 associate with schizophrenia, our studies identify a biochemical pathway important for brain development and mental health.

Myelin development, plasticity, and pathology in the auditory system.

Myelin allows for the rapid and precise timing of action potential propagation along neuronal circuits and is essential for healthy auditory system function. In this article, we discuss what is currently known about myelin in the auditory system with a focus on the timing of myelination during auditory system development, the role of myelin in supporting peripheral and central auditory circuit function, and how various myelin pathologies compromise auditory information processing. Additionally, in keeping with the increasing recognition that myelin is dynamic and is influenced by experience throughout life, we review the growing evidence that auditory sensory deprivation alters myelin along specific segments of the brain's auditory circuit. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 78: 80-92, 2018.

From Otic Induction to Hair Cell Production: Pax2EGFP Cell Line Illuminates Key Stages of Development in Mouse Inner Ear Organoid Model.

Producing hair cells of the inner ear is the major goal of ongoing research that combines advances in developmental and stem cell biology. The recent advent of an inner ear organoid protocol-resulting in three-dimensional stem cell-derived tissues resembling vestibular sensory epithelia-has sparked interest in applications such as regeneration, drug discovery, and disease modeling. In this study, we adapted this protocol for a novel mouse embryonic stem cell line with a fluorescent reporter for Pax2 expression. We used Pax2EGFP/+ organoid formation to model otic induction, the pivotal developmental event when preplacodal tissue adopts otic fate. We found upregulation of Pax2 and activation of ERK downstream of fibroblast growth factor signaling in organoid formation as in embryonic inner ear development. Pax2 expression was evident from the EGFP reporter beginning at the vesicle formation stage and persisting through generation of the sensory epithelium. The native ventralizing signal sonic hedgehog was largely absent from the cell aggregates as otic vesicles began to form, confirming the dorsal vestibular organoid fate. Nonetheless, cochlear- or vestibular-like neurons appeared to delaminate from the derived otic vesicles and formed synaptic contacts with hair cells in the organoids. Cell lines with transcriptional reporters such as Pax2EGFP/+ facilitate direct evaluation of morphological changes during organoid production, a major asset when establishing and validating the culture protocol.

Transient auditory nerve demyelination as a new mechanism for hidden hearing loss.

Hidden hearing loss (HHL) is a recently described auditory neuropathy believed to contribute to speech discrimination and intelligibility deficits in people with normal audiological tests. Animals and humans with HHL have normal auditory thresholds but defective cochlear neurotransmission, that is, reduced suprathreshold amplitude of the sound-evoked auditory nerve compound action potential. Currently, the only cellular mechanism known for HHL is loss of inner hair cell synapses (synaptopathy). Here we report that transient loss of cochlear Schwann cells results in permanent auditory deficits characteristic of HHL. This auditory neuropathy is not associated with synaptic loss, but rather with disruption of the first heminodes at the auditory nerve peripheral terminal. Thus, this study identifies a new mechanism for HHL, highlights the long-term consequences of transient Schwann cell loss on hearing and might provide insights into the causes of the auditory deficits reported in patients that recover from acute demyelinating diseases such as Guillain-Barré syndrome.

Transcriptional Regulation of lncRNA Genes by Histone Modification in Alzheimer's Disease.

Increasing studies have revealed that long noncoding RNAs (lncRNAs) are not transcriptional noise but play important roles in the regulation of a wide range of biological processes, and the dysregulation of lncRNA genes is associated with disease development. Alzheimer's disease (AD) is a chronic neurodegenerative disease that usually starts slowly and gets worse over time. However, little is known about the roles of lncRNA genes in AD and how the lncRNA genes are transcriptionally regulated. Herein, we analyzed RNA-seq data and ChIP-seq histone modification data from CK-p25 AD model and control mice and identified 72 differentially expressed lncRNA genes, 4,917 differential peaks of H3K4me3, and 1,624 differential peaks of H3K27me3 between AD and control samples, respectively. Furthermore, we found 92 differential peaks of histone modification H3K4me3 are located in the promoter of 39 differentially expressed lncRNA genes and 8 differential peaks of histone modification H3K27me3 are located upstream of 7 differentially expressed lncRNA genes, which suggest that the majority of lncRNA genes may be transcriptionally regulated by histone modification in AD.

No longer falling on deaf ears: mechanisms of degeneration and regeneration of cochlear ribbon synapses.

Cochlear ribbon synapses are required for the rapid and precise neural transmission of acoustic signals from inner hair cells to the spiral ganglion neurons. Emerging evidence suggests that damage to these synapses represents an important form of cochlear neuropathy that might be highly prevalent in sensorineural hearing loss. In this review, we discuss our current knowledge on how ribbon synapses are damaged by noise and during aging, as well as potential strategies to promote ribbon synapse regeneration for hearing restoration.