TNN is first linked to auditory neuropathy.

Autosomal recessive nonsyndromic auditory neuropathy is attributed to a genetic etiology. We identified a compound heterozygous missense variant, c.G736A (p.G246S) and c.C2954T (p.T985 M) in TNN of affected patients in a pedigree via candidate gene screening and exome sequencing. To determine the genetic etiology of deafness in the pedigree with a heterozygous missense variant in the gene TNN encoding tenascin-W associated with autosomal recessive nonsyndromic auditory neuropathy, the cochlear expression of tenascin-W was evaluated at mRNA and protein levels in mice, and Tnn knock out mice were generated and utilized to study the function of Tnn in the auditory system. Immunofluorescence stainings showed that tenascin-W was mainly expressed in the somatic cytoplasm of spiral ganglion neurons of mice. Homozygous Tnn knockout was lethal in mice, whereas Tnn heterozygous mice showed decreases in spiral ganglion neuron density and progressive hearing loss. We demonstrate that tenascin-W is expressed in the murine cochleae and is essential for the development of spiral ganglion neurons. An abnormal expression of tenascin-W can influence the development and function of SGNs and affect the function of the auditory system.

The pyroptosis mechanism of ototoxicity caused by unconjugated bilirubin in neonatal hyperbilirubinemia.

When activated by unconjugated bilirubin (UCB), inflammatory mediators such as IL - 18 and TNF contribute to the neurotoxicity and ototoxicity observed in severe neonatal hyperbilirubinemia. However, in cell and molecular level, the regulation and mechanism of UCB-induced ototoxicity are remained unclear. In this study, 7-day-old mammary rats were exposed to various concentrations of UCB to imitate the infant auditory damage. The auditory brainstem response result (ABR) indicated severe hearing loss, which occurred with increasing concentration. Morphological analysis of organotypic cochlear cultures treated with different concentrations of UCB indicated that auditory nerve fibers (ANF) were demyelinated and the density of spiral ganglion neurons (SGN) were decreased. In addition, HEI-OC1 cells treated with different concentrations of UCB showed severe necrosis by Flow Cytometry. The morphologic feature of pyroptosis has been observed by scanning electronic microscope. Cleaved Caspase-1, GSDMD and NLRP3 expression were significantly increased in cochlear explants with UCB-induced. To further clarify the molecular mechanism of UCB-induced inner ear cell pyroptosis, specific inhibitors of pyroptosis were applied, the protein associated with pyrotosis such as Cleaved Caspase-1, GSDMD, ASC, IL-18 and NLRP3 were significantly lower than the group with UCB alone. All the data above indicated that ERK /NLRP3/GSDMD signaling pathway involved in UCB-induced ototoxicity.

Sex differences in glutamate AMPA receptor subunits mRNA with fast gating kinetics in the mouse cochlea.

Evidence shows that females have increased supra-threshold peripheral auditory processing compared to males. This is indicated by larger auditory brainstem responses (ABR) wave I amplitude, which measures afferent spiral ganglion neuron (SGN)-auditory nerve synchrony. However, the underlying molecular mechanisms of this sex difference are mostly unknown. We sought to elucidate sex differences in ABR wave I amplitude by examining molecular markers known to affect synaptic transmission kinetics. Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) mediate fast excitatory transmission in mature SGN afferent synapses. Each AMPAR channel is a tetramer composed of GluA2, 3, and 4 subunits (Gria2, 3, and 4 genes), and those lacking GluA2 subunits have larger currents, are calcium-permeable, and have faster gating kinetics. Moreover, alternatively spliced flip and flop isoforms of each AMPAR subunit affect channel kinetics, having faster kinetics those AMPARs containing Gria3 and Gria4 flop isoforms. We hypothesized that SGNs of females have more fast-gating AMPAR subunit mRNA than males, which could contribute to more temporally precise synaptic transmission and increased SGN synchrony. Our data show that the index of Gria3 relative to Gria2 transcripts on SGN was higher in females than males (females: 48%; males: 43%), suggesting that females have more SGNs with higher Gria3 mRNA relative to Gria2. Analysis of the relative abundance of the flip and flop alternatively spliced isoforms showed that females have a 2-fold increase in fast-gating Gria3 flop mRNA, while males have more slow-gating (2.5-fold) of the flip. We propose that Gria3 may in part mediate greater SGN synchrony in females. Significance Statement: Females of multiple vertebrate species, including fish and mammals, have been reported to have enhanced sound-evoked synchrony of afferents in the auditory nerve. However, the underlying molecular mediators of this physiologic sex difference are unknown. Elucidating potential molecular mechanisms related to sex differences in auditory processing is important for maintaining healthy ears and developing potential treatments for hearing loss in both sexes. This study found that females have a 2-fold increase in Gria3 flop mRNA, a fast-gating AMPA-type glutamate receptor subunit. This difference may contribute to greater neural synchrony in the auditory nerve of female mice compared to males, and this sex difference may be conserved in all vertebrates.

Inhibiting DNA methylation alleviates cisplatin-induced hearing loss by decreasing oxidative stress-induced mitochondria-dependent apoptosis via the LRP1-PI3K/AKT pathway.

Cisplatin-related ototoxicity is a critical side effect of chemotherapy and can lead to irreversible hearing loss. This study aimed to assess the potential effect of the DNA methyltransferase (DNMT) inhibitor RG108 on cisplatin-induced ototoxicity. Immunohistochemistry, apoptosis assay, and auditory brainstem response (ABR) were employed to determine the impacts of RG108 on cisplatin-induced injury in murine hair cells (HCs) and spiral ganglion neurons (SGNs). Rhodamine 123 and TMRM were utilized for mitochondrial membrane potential (MMP) assessment. Reactive oxygen species (ROS) amounts were evaluated by Cellrox green and Mitosox-red probes. Mitochondrial respiratory function evaluation was performed by determining oxygen consumption rates (OCRs). The results showed that RG108 can markedly reduce cisplatin induced damage in HCs and SGNs, and alleviate apoptotic rate by protecting mitochondrial function through preventing ROS accumulation. Furthermore, RG108 upregulated BCL-2 and downregulated APAF1, BAX, and BAD in HEI-OC1 cells, and triggered the PI3K/AKT pathway. Decreased expression of low-density lipoprotein receptor-related protein 1 (LRP1) and high methylation of the LRP1 promoter were observed after cisplatin treatment. RG108 treatment can increase LRP1 expression and decrease LRP1 promoter methylation. In conclusion, RG108 might represent a new potential agent for preventing hearing loss induced by cisplatin via activating the LRP1-PI3K/AKT pathway.

A dual metal organic framework based on copper-iron clusters integrated sulphur doped graphene as a porous material for supercapacitor with remarkable performance characteristics.

Herein, a novel bimetallic metal organic framework (MOF) using copper and iron as the metal centers with 1,3,5-tricarboxylic acid as a ligand (CuFeBTC) and its composite with sulphur doped graphene (S-GNS) have been investigated for supercapacitive performance. The synthesis of materials has been carried out using a facile wet chemical route. The physicochemical characterization of the materials employing various structural and surface techniques has been performed which confirms the successful formation of nanocomposite. The capacitive behavior of CuFeBTC, S-GNS and CuFeBTC/S-GNS has been systematically examined using 1 M Na2SO4 as an electrolyte in a three and two electrode assembly. The electrochemical studies reveal that CuFeBTC/S-GNS electrode demonstrates highest specific capacitance of 1164.3 F g-1 at 0.5 A g-1 with suffice rate capability as compared to CuFeBTC and S-GNS electrodes. Moreover, a symmetric supercapacitor is configured using the CuFeBTC/S-GNS nanocomposite electrodes which deliver remarkable energy and power output of 96.57 Wh kg-1 and 1595.12 W kg-1 at an operating voltage of 1.8 V. The as-fabricated symmetric supercapacitor displays competent energy storage retention of 50.2 Wh kg-1 even at current density of 20.0 A g-1 with high power density 26973.13 W kg-1. These deliverables epitomize the latest performance record of bimetallic MOFs based supercapacitors, suggesting that CuFeBTC/S-GNS is a promising active material for high performance electrochemical energy storage applications.

Increased levels of miR-124 in human dental pulp stem cells alter the expression of neural markers.

Auditory neuropathy is the particular form of deafness in humans which cannot be treated by replacement therapy. Human dental pulp stem cells (hDPSCs) are derived from an ectomesenchymal neural crest cell population. Therefore, they possess a promising capacity for neuronal differentiation and repair. miR-124, a key regulator of neuronal development in the inner ear, is expressed at high levels in auditory and vestibular neurons. Here, we evaluated the possible effect of miR-124 in alteration of neural protein markers expression. Using quantitative reverse transcription-PCR (qRT-PCR) analyses and immunofluorescence staining, we studied the expression patterns of neural progenitor markers (Nestin, NOTCH1, and SOX2) and neural markers (ß-tubulin III, GATA-3, and peripherin) upon transfection of hDPSCs with miR-124. The qRT-PCR results showed that Nestin was upregulated 6 h post-transfection. In contrast, Nestin expression exhibited a decreasing trend 24 h and 48 h post-transfection. Higher levels of ß-tubulin III, 6 h and 16 h post transfection in RNA level as compared with control cells, were determined in transfected DPSCs. However, ß-tubulin-III expression decreased 48 h post-transfection. The immunoflourescence results indicated that transfection of hDPSCs with miR-124, only affected Nestin among the studied neural progenitor and neural marker expression in protein level.

Schwann cells genetically modified to express S100A4 increases GAP43 expression in spiral ganglion neurons in vitro.

Schwann cells (SCs) have been reported as a possible source of neurotrophic support for spiral ganglion neurons (SGNs). This study was aimed to investigate whether S100A4 was contributed in the functional effects of SCs on SGNs. SCs were transfected with S100A4 vector or small interfering RNA (siRNA) against S100A4, and the transfection efficiency was verified by quantitative PCR (qPCR) and Western blot. The migration of transfected SCs was determined by Transwell assay, and the expression levels of vascular endothelial growth factor precursor (VEGF) and matrix metallopeptidase 9 (MMP-9) were measured by Western blot. Co-culture of either S100A4 overexpressed or suppressed SCs with SGNs, and the growth associated protein 43 (GAP43) expression in SGNs was detected by immunofluorescence (IF), qPCR and Western blot. The migration of SCs was significantly enhanced by S100A4 overexpression (P < 0.001), while was suppressed by S100A4 knockdown (P < 0.01). Further, the expressions of VEGF and MMP-9 were notably up-regulated by S100A4 overexpression, while were down-regulated by S100A4 knockdown. Moreover, co-culture with the S100A4 overexpressed SCs significantly increased the expression of GAP43 in SGNs (P < 0.01). As expected, co-culture with S100A4 knockdown SCs decreased GAP43 level (P < 0.05). S100A4 enhanced the migratory ability of SCs. SCs genetically modified to overexpress the S100A4 could up-regulate the GAP43 expression in SGNs.

In Vitro Functional Assessment of Adult Spiral Ganglion Neurons (SGNs).

Spiral ganglion neurons (SGNs) faithfully encode acoustic waves from hair cells to the cochlear nucleus (CN) using voltage-dependent ion channels. A sizable portion of our knowledge on SGN functions have been derived from pre-hearing neurons. In post-hearing SGNs, the mechanisms of how they encode the massive sound information without delay and precisely are largely unknown. Mature SGNs are housed in the central bony labyrinth of the cochlea, protected by a well-insulated myelin sheath, making it a technical feat to isolate viable neurons for rigorous functional electrophysiology. Recently, we have overcome the previous intractable hindrance in SGN functional analyses. We provide a step-by-step user-friendly protocol with practical applications, including patch-clamp recordings and imaging by using cultured SGNs.

Topographic and quantitative evaluation of gentamicin-induced damage to peripheral innervation of mouse cochleae.

Ototoxicity induced by aminoglycoside antibiotics appears to occur both in hair cells (HCs) and the cochlear nerves that innervate them. Although HC loss can be easily quantified, neuronal lesions are difficult to quantify because two types of afferent dendrites and two types of efferent axons are tangled beneath the hair cells. In the present study, ototoxicity was induced by gentamicin in combination with the diuretic agent furosemide. Neuronal lesions were quantified in cochlear whole-mount preparations combined with microsections across the habenular perforate (HP) openings to achieve a clear picture of the topographic relationship between neuronal damage and HC loss. Multiple immunostaining methods were employed to differentiate the two types of afferent dendrites and two types of efferent axons. The results show that co-administration of gentamicin and furosemide resulted in a typical dynamic pattern of HC loss that spread from the basal turn to the outer hair cells to the apex and inner hair cells, depending on the dose and survival time after drug administration. Lesions of the innervation appeared to occur at two stages. At the early stage (2-4 days), the loss of labeling of the two types of afferent dendrites was more obvious than the loss of labeled efferent axons. At the late stage (2-4 weeks), the loss of labeled efferent axons was more rapid. In the high-dose gentamicin group, the loss of outer HCs was congruent with afferent dendrite loss at the early stage and efferent axon loss at the late stage. In the low-dose gentamicin group, the loss of labeling for cochlear innervation was more severe and widespread. Thus, we hypothesize that the gentamicin-induced damage to cochlear innervation occurs independently of hair cell loss.