The p. S178L mutation in Tbc1d24 disrupts endosome-mediated synaptic vesicle trafficking of cochlear hair cells and leads to hearing impairment in mice.

The ribbon synapses of cochlear inner hair cells (IHCs) employ efficient vesicle resupply to enable fast and sustained release rates. However, the molecular mechanisms of these physiological activities remain unelucidated. Previous studies showed that the RAB-specific GTPase-activating protein TBC1D24 controls the endosomal trafficking of the synaptic vesicles (SVs) in Drosophila and mammalian neurons, and mutations in TBC1D24 may lead to non-syndromic hearing loss or hearing loss associated with the DOORS syndrome in humans. In this study, we generated a knock-in mouse model for the p. S178L mutation in TBC1D24, which leads to autosomal dominant non-syndromic hearing loss (DFNA65). The p.S178L mutant mice show mild hearing loss and progressively declined wave I amplitude of the auditory brainstem responses. Despite the normal gross and cellular morphology of the cochlea, transmission electron microscopy reveals accumulation of endosome-like vacuoles and a lower-than-normal number of SVs directly associated with the ribbons in the IHCs. Consistently, patch clamp of the IHCs shows reduced exocytosis under prolonged stimulus. ARF6, a TBC1D24-interacting protein also involved in endosomal membrane trafficking, was underexpressed in the cochleae of the mutant mouse and has weakened in vitro interaction with the p.S178L mutant TBC1D24. Our results suggest an important role of TBC1D24 in maintaining endosomal-mediated vesicle recycling and sustained exocytosis of hair cell ribbon synapses.

Connexin30-deficient mice increase susceptibility to noise via redox and lactate imbalances.

Noise significantly contributes to one-third of the global burden of hearing loss. The intricate interplay of genetic and environmental factors impacts various molecular and cellular processes that lead to noise-induced hearing loss (NIHL). Defective connexin 26 (Cx26) and connexin 30 (Cx30), encoded by Gjb2/Cx26 and Gjb6/Cx30, respectively, are prevalent causes of hereditary deafness. However, the role of Cx30 in the pathogenesis of NIHL remains unclear. Herein, we observed that homozygous Cx30 knockout (Cx30 KO) mice exhibited poorer hearing recovery after noise exposure (97 dB mean sound pressure level for 2 h) and increased susceptibility to noise. In addition to the exacerbation of noise-induced damage to hair cells and synapses, Cx30 KO mice exposed to noise exhibited increased oxidative stress. The 2-(N-(7-nitrobenz-2-oxa-1,3-dia-zol-4-yl) amino)-2-deoxyglucose assay showed a reduction in glucose levels associated with a decrease in gap junctions as well as a reduction in adenosine triphosphate release. Glucose metabolomics analysis further revealed that Cx30 KO mice had elevated lactate and NAD+ levels after noise exposure, thus worsening anaerobic oxidation from glycolysis. Our study emphasizes that Cx30-deficient mice increase susceptibility to noise via redox and lactate imbalances in the cochlea.

Pathological mechanisms of connexin26-related hearing loss: Potassium recycling, ATP-calcium signaling, or energy supply?

Hereditary deafness is one of the most common human birth defects. GJB2 gene mutation is the most genetic etiology. Gap junction protein 26 (connexin26, Cx26) encoded by the GJB2 gene, which is responsible for intercellular substance transfer and signal communication, plays a critical role in hearing acquisition and maintenance. The auditory character of different Connexin26 transgenic mice models can be classified into two types: profound congenital deafness and late-onset progressive hearing loss. Recent studies demonstrated that there are pathological changes including endocochlear potential reduction, active cochlear amplification impairment, cochlear developmental disorders, and so on, in connexin26 deficiency mice. Here, this review summarizes three main hypotheses to explain pathological mechanisms of connexin26-related hearing loss: potassium recycling disruption, adenosine-triphosphate-calcium signaling propagation disruption, and energy supply dysfunction. Elucidating pathological mechanisms underlying connexin26-related hearing loss can help develop new protective and therapeutic strategies for this common deafness. It is worthy of further study on the detailed cellular and molecular upstream mechanisms to modify connexin (channel) function.

Dietary intake of deuterium oxide decreases cochlear metabolism and oxidative stress levels in a mouse model of age-related hearing loss.

Age-related hearing loss (ARHL) is the most prevalent sensory disorder in the elderly. Currently, no treatment can effectively prevent or reverse ARHL. Aging auditory organs are often accompanied by exacerbated oxidative stress and metabolic deterioration. Here, we report the effect of deuterated oxygen (D2O), also known as "heavy water", mouse models of ARHL. Supplementing the normal mouse diet with 10% D2O from 4 to 9 weeks of age lowered hearing thresholds at selected frequencies in treated mice compared to untreated control group. Oxidative stress levels were significantly reduced and in the cochlear duct of treated vs. untreated mice. Through metabolic flux analysis, we found that D2O mainly slowed down catabolic reactions, and may delay metabolic deterioration related to aging to a certain extent. Experiments confirmed that the Nrf2/HO-1/glutathione axis was down-regulated in treated mice. Thus, D2O supplementation can hinder ARHL progression in mouse models by slowing the pace of metabolism and reducing endogenous oxidative stress production in the cochlea. These findings open new avenues for protecting the cochlea from oxidative stress and regulating metabolism to prevent ARHL.

FGF22 deletion causes hidden hearing loss by affecting the function of inner hair cell ribbon synapses.

Ribbon synapses are important structures in transmitting auditory signals from the inner hair cells (IHCs) to their corresponding spiral ganglion neurons (SGNs). Over the last few decades, deafness has been primarily attributed to the deterioration of cochlear hair cells rather than ribbon synapses. Hearing dysfunction that cannot be detected by the hearing threshold is defined as hidden hearing loss (HHL). The relationship between ribbon synapses and FGF22 deletion remains unknown. In this study, we used a 6-week-old FGF22 knockout mice model (Fgf22 -/-) and mainly focused on alteration in ribbon synapses by applying the auditory brainstem response (ABR) test, the immunofluorescence staining, the patch-clamp recording, and quantitative real-time PCR. In Fgf22 -/- mice, we found the decreased amplitude of ABR wave I, the reduced vesicles of ribbon synapses, and the decreased efficiency of exocytosis, which was suggested by a decrease in the capacitance change. Quantitative real-time PCR revealed that Fgf22 - / - led to dysfunction in ribbon synapses by downregulating SNAP-25 and Gipc3 and upregulating MEF2D expression, which was important for the maintenance of ribbon synapses' function. Our research concluded that FGF22 deletion caused HHL by affecting the function of IHC ribbon synapses and may offer a novel therapeutic target to meet an ever-growing demand for deafness treatment.

Pseudo-Temporal Analysis of Single-Cell RNA Sequencing Reveals Trans-Differentiation Potential of Greater Epithelial Ridge Cells Into Hair Cells During Postnatal Development of Cochlea in Rats.

The hair cells of the cochlea play a decisive role in the process of hearing damage and recovery, yet knowledge of their regeneration process is still limited. Greater epithelial ridge (GER) cells, a type of cell present during cochlear development that has the characteristics of a precursor sensory cell, disappear at the time of maturation of hearing development. Its development and evolution remain mysterious for many years. Here, we performed single-cell RNA sequencing to profile the gene expression landscapes of rats' cochlear basal membrane from P1, P7, and P14 and identified eight major subtypes of GER cells. Furthermore, single-cell trajectory analysis for GER cells and hair cells indicated that among the different subtypes of GER, four subtypes had transient cell proliferation after birth and could transdifferentiate into inner and outer hair cells, and two of them mainly transdifferentiated into inner hair cells. The other two subtypes eventually transdifferentiate into outer hair cells. Our study lays the groundwork for elucidating the mechanisms of the key regulatory genes and signaling pathways in the trans-differentiation of GER cell subtypes into hair cells and provides potential clues to understand hair cell regeneration.

Failure Of Hearing Acquisition in Mice With Reduced Expression of Connexin 26 Correlates With the Abnormal Phasing of Apoptosis Relative to Autophagy and Defective ATP-Dependent Ca2+ Signaling in Kölliker's Organ.

Mutations in the GJB2 gene that encodes connexin 26 (Cx26) are the predominant cause of prelingual hereditary deafness, and the most frequently encountered variants cause complete loss of protein function. To investigate how Cx26 deficiency induces deafness, we examined the levels of apoptosis and autophagy in Gjb2 loxP/loxP; ROSA26 CreER mice injected with tamoxifen on the day of birth. After weaning, these mice exhibited severe hearing impairment and reduced Cx26 expression in the cochlear duct. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positive cells were observed in apical, middle, and basal turns of Kölliker's organ at postnatal (P) day 1 (P1), associated with increased expression levels of cleaved caspase 3, but decreased levels of autophagy-related proteins LC3-II, P62, and Beclin1. In Kölliker's organ cells with decreased Cx26 expression, we also found significantly reduced levels of intracellular ATP and hampered Ca2+ responses evoked by extracellular ATP application. These results offer novel insight into the mechanisms that prevent hearing acquisition in mouse models of non-syndromic hearing impairment due to Cx26 loss of function.

Overexpression of XIAP inhibits cisplatin-induced hair cell loss.

Cisplatin is a platinum-containing drug with ototoxicity commonly used clinically and has significant efficacy against a variety of solid tumors. One of the most important mechanisms of ototoxicity is that cisplatin induces apoptosis of hair cells. According to relevant literature, X-linked inhibitor of apoptosis protein (XIAP, anti-apoptotic protein) could inhibit the apoptotic pathway. We hypothesized that this protein might protect cochlear hair cells from cisplatin-induced injury. To figure it out, we treated cochlea of normal mice with various concentrations of cisplatin to observe the response and morphology of hair cells and determine a reasonable concentration. Next, Western Blot and quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) experiments were conducted to make an investigation about the expression of XIAP protein and mRNA. In addition, we constructed and identified XIAP overexpressing mice. Finally, we treated cochlear tissues of normal and overexpressing mice with cisplatin to investigate the cyto-protection of XIAP on hair cells, respectively. It was found that 50 µmol/L cisplatin resulted in significant loss and disorganization of hair cells, while simultaneously downregulating the protein and mRNA of XIAP. In XIAP overexpressing mice, the loss and disorganization of hair cells were significantly lessened. These results showed that XIAP can lessen cisplatin-induced hair cell loss and play a role in otoprotection.

Single-Cell RNA Sequencing Analysis Reveals Greater Epithelial Ridge Cells Degeneration During Postnatal Development of Cochlea in Rats.

Greater epithelial ridge cells, a transient neonatal cell group in the cochlear duct, which plays a crucial role in the functional maturation of hair cell, structural development of tectorial membrane, and refinement of audio localization before hearing. Greater epithelial ridge cells are methodologically homogeneous, while whether different cell subtypes are existence in this intriguing region and the degeneration mechanism during postnatal cochlear development are poorly understood. In the present study, single-cell RNA sequencing was performed on the cochlear duct of postnatal rats at day 1 (P1) and day 7 (P7) to identify subsets of greater epithelial ridge cell and progression. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were used to examine genes enriched biological processes in these clusters. We identified a total of 26 clusters at P1 and P7 rats and found that the cell number of five cell clusters decreased significantly, while four clusters had similar gene expression patterns and biological properties. The genes of these four cell populations were mainly enriched in Ribosome and P13K-Akt signal pathway. Among them, Rps16, Rpsa, Col4a2, Col6a2, Ctsk, and Jun are particularly interesting as their expression might contribute to the greater epithelial ridge cells degeneration. In conclusion, our study provides an important reference resource of greater epithelial ridge cells landscape and mechanism insights for further understanding greater epithelial ridge cells degeneration during postnatal rat cochlear development.

Hearing Protection Outcomes of Analog Electrode Arrays Coated with Different Drug-Eluting Polymer Films Implanted into Guinea Pig Cochleae.

OBJECTIVE: To investigate the hearing protection outcomes of different drug-eluting analog electrode arrays implanted into guinea pig cochleae. METHODS: Sixty guinea pigs were randomly divided into a negative control group and five experimental groups implanted separately with blank (drug carrier), dexamethasone (DXM), aracytine (Ara-C), Ara-C+DXM, and nicotinamide adenine dinucleotide (NAD+) eluting analog electrode arrays. Micro CT was used to supervise the surgical procedure. Auditory brainstem response (ABR) thresholds of the guinea pigs were measured and analyzed. RESULTS AND CONCLUSIONS: Compared with the negative control, all other groups showed a significant increase in ABR threshold (p<0.001) after surgery. Among them, there was no obvious difference between the blank (0 vs 90 days: 59.70±10.57 vs 64.60±9.47 dB SPL) and the NAD+ group (0 vs 90 days: 59.90±9.87 vs 64.70±8.65 dB SPL). On the other hand, the ABR thresholds in the DXM (0 days: 58.10±10.73 dB SPL; 90 days: 51.70±9.07 dB SPL) and the Ara-C group (0 days: 59.00±10.05 dB SPL; 90 days: 51.60±8.48 dB SPL) decreased significantly compared with the former two groups (p<0.001). However, the Ara-C+DXM group showed no further benefit (p>0.05). In addition, a significantly higher survival rate of spiral ganglion neurons in cochleae was observed in the Ara-C and/or DXM groups.

Connexin30-Deficiency Causes Mild Hearing Loss With the Reduction of Endocochlear Potential and ATP Release.

GJB2 and GJB6 are adjacent genes encoding connexin 26 (Cx26) and connexin 30 (Cx30), respectively, with overlapping expressions in the inner ear. Both genes are associated with the commonest monogenic hearing disorder, recessive isolated deafness DFNB1. Cx26 plays an important role in auditory development, while the role of Cx30 in hearing remains controversial. Previous studies found that Cx30 knockout mice had severe hearing loss along with a 90% reduction in Cx26, while another Cx30 knockout mouse model showed normal hearing with nearly half of Cx26 preserved. In this study, we used CRISPR/Cas9 technology to establish a new Cx30 knockout mouse model (Cx30-/-), which preserves approximately 70% of Cx26. We found that the 1, 3, and 6-month-old Cx30-/- mice showed mild hearing loss at full frequency. Immunofluorescence and HE staining suggested no significant differences in microstructure of the cochlea between Cx30-/- mice and wild-type mice. However, transmission electron microscopy showed slight cavity-like damage in the stria vascularis of Cx30-/- mice. And Cx30 deficiency reduced the production of endocochlear potential (EP) and the release of ATP, which may have induced hearing loss. Taken together, this study showed that lack of Cx30 can lead to hearing loss with an approximately 30% reduction of Cx26 in the present Cx30 knockout model. Hence, Cx30 may play an important rather than redundant role in hearing development.

Distinct Expression Patterns of Apoptosis and Autophagy-Associated Proteins and Genes during Postnatal Development of Spiral Ganglion Neurons in Rat.

Autophagy and apoptosis have a complex interplay in the early embryo development. The development of spiral ganglion neurons (SGNs) in addition to Corti's organ in the mammalian cochlea remains crucial in the first two-week postnatal period. To investigate the roles of apoptosis and autophagy in the development of SGNs, light microscopy was used to observe the morphological changes of SGNs. The number of SGNs was decreased from P1 to P7 and plateaued from P10 to P14. Immunohistochemistry results revealed positive expression of cleaved-caspase3, bcl-2, microtubule-associated protein light chain 3-II (LC3-II), Beclin1, and sequestosome 1 (SQSTM1/P62) in SGNs. The apoptotic bodies and autophagosomes and autolysosomes were also identified by transmission electron microscopy at P1 and P7. Real-time PCR and western blotting results revealed that the apoptotic activity peaked at P7 and the autophagy activity was gradually upregulated along with the development. Taken together, our results for the first time showed that autophagy and apoptosis in SGNs play distinct roles during specific developmental phases in a time-dependent manner.

Identification of MYO6 copy number variation associated with cochlear aplasia by targeted sequencing.

Copy number variation is an extensively studied cause of hereditary diseases. However, its role in hereditary sensorineural deafness has been rarely reported. Using targeted sequencing, SNP array and qPCR, we found a novel 622.2 kb duplication of 6q14.1 in a patient with congenital sensorineural hearing loss and cochlear aplasia. The duplication included MYO6 and IMPG1 genes. FISH study confirmed that this duplication was inherited from the patient's mosaic mother.

Management of Subclinical and Overt Hypothyroidism Following Hemithyroidectomy in Children and Adolescents: A Pilot Study.

Background: To reduce surgical complications and avoid lifelong thyroid hormone replacement, hemithyroidectomy is preferred in children and adolescents with benign nodular thyroid disease. However, hypothyroidism following hemithyroidectomy may occur, and postoperative thyroid hormone replacement for hypothyroidism following hemithyroidectomy is usually administered without a full understanding of the clinical characteristics of hypothyroidism. Methods: To investigate the incidence and risk factors of hypothyroidism after hemithyroidectomy in children and adolescents, and to identify whether postoperative thyroid hormone replacement is necessary, a retrospective review of 43 patients under 18 years of age who underwent hemithyroidectomy from January 2009 to October 2016 was conducted. All hypothyroid patients were retrospectively analyzed to determine the incidence and predisposing factor(s) of postoperative hypothyroidism. All patients were measured regarding age, sex, serum thyrotropin (TSH), anti-thyroid antibody, and histological evidence of lymphocytic infiltration. Hypothyroid patients were measured for symptoms, timing of diagnosis, and thyroid hormone replacement. Results: The mean age at the time of surgery was 13.65 ± 3.04 years. Of the cohort, 34 patients were female (79.07%), and the mean follow-up time was 28 ± 9 months. Hypothyroidism was diagnosed in 11 of the 43 patients. The mean postoperative TSH level was 7.17 ± 2.13 µIU/ml. The mean preoperative TSH level was 3.11 ± 0.59 µIU/ml in hypothyroid patients compared with 1.92 ± 0.72 µIU/ml in euthyroid patients (P < 0.05). A preoperative TSH level >2.2 µIU/l and lymphocytic infiltration graded 3 or 4 were found to be independent risk factors for the development of hypothyroidism. There were no significant differences between groups in terms of patient age or sex. Conclusions: In the pediatric and adolescent population, patients with elevated preoperative TSH levels or the presence of lymphocytic infiltration may increase the risk of risk of hypothyroidism. In our study, postoperative levothyroxine (L-T4) treatment was necessary in 16.28% of cases after hemithyroidectomy. Patients with mild postoperative hypothyroidism should be followed up, without the need for immediate L-T4 replacement, so as to expect patients to recover spontaneously.

ATP is stored in lysosomes of greater epithelial ridge supporting cells in newborn rat cochleae.

Adenosine triphosphate (ATP), which plays a crucial role in both developing and mature cochleae, is released from greater epithelial ridge (GER) supporting cells of the rat cochlea, but the organelles in which ATP is stored have not yet been identified. Thus, we studied the organelles involved in ATP storage and suggest that lysosomes provide this function. GER supporting cells of newborn rats were isolated, purified, and cultured, and labeled vesicles within the supporting cells were identified via confocal microscopy and transmission electron microscopy (TEM). ATP release from GER supporting cells after glycyl-L-phenylalanine-ß-naphthylamide (GPN) treatment was measured. The specifically labeled organelles observed by confocal microscopy and TEM were lysosomes, and GPN treatment enhanced ATP luminescence in the extracellular fluid of the supporting cells. The release of ATP from supporting cells was affected by changes in intra- and extracellular Ca2+ concentrations. In addition, changes in the intracellular Ca2+ caused by inhibiting the phospholipase signaling pathway affected the release of ATP from supporting cells. We demonstrated that ATP is stored in the lysosomes of GER supporting cells within newborn rat cochleae and that ATP release from GER supporting cells may be Ca2+ -dependent.

Autophagy precedes apoptosis during degeneration of the Kölliker's organ in the development of rat cochlea.

The Kölliker's organ is a transient epithelial structure during cochlea development that gradually degenerates and disappears at postnatal 12-14 days (P12-14). While apoptosis has been shown to play an essential role in the degeneration of the Kölliker's organ, the role of another programmed cell death, autophagy, remains unclear. In our study, autophagy markers including microtubule associated protein light chain 3-II (LC3-II), sequestosome 1 (SQSTM1/p62) and Beclin1 were detected in the supporting cells of the Kölliker's organ through immunohistochemistry staining. In addition, Western blot and real-time PCR revealed a gradually decreased expression of LC3-II and an increased expression of p62 during early postnatal development. Compared to apoptosis markers that peaks between P7 and P10, autophagy flux peaked earlier at P1 and decreased from P1 to P14. By transmission electron microscopy, we observed representative autophagosome and autolysosome that packaged various organelles in the supporting cells of the Kölliker's organ. During the degeneration, these organelles were digested via autophagy well ahead of the cellular apoptosis. These results suggest that autophagy plays an important role in transition and degeneration of the Kölliker's organ prior to apoptosis during the early postnatal development.