Metabolic Profiling of Cochlear Organoids Identifies α-Ketoglutarate and NAD+ as Limiting Factors for Hair Cell Reprogramming.

Cochlear hair cells are the sensory cells responsible for transduction of acoustic signals. In mammals, damaged hair cells do not regenerate, resulting in permanent hearing loss. Reprogramming of the surrounding supporting cells to functional hair cells represent a novel strategy to hearing restoration. However, cellular processes governing the efficient and functional hair cell reprogramming are not completely understood. Employing the mouse cochlear organoid system, detailed metabolomic characterizations of the expanding and differentiating organoids are performed. It is found that hair cell differentiation is associated with increased mitochondrial electron transport chain (ETC) activity and reactive oxidative species generation. Transcriptome and metabolome analyses indicate reduced expression of oxidoreductases and tricyclic acid (TCA) cycle metabolites. The metabolic decoupling between ETC and TCA cycle limits the availability of the key metabolic cofactors, α-ketoglutarate (α-KG) and nicotinamide adenine dinucleotide (NAD+). Reduced expression of NAD+ in cochlear supporting cells by PGC1α deficiency further impairs hair cell reprogramming, while supplementation of α-KG and NAD+ promotes hair cell reprogramming both in vitro and in vivo. These findings reveal metabolic rewiring as a central cellular process during hair cell differentiation, and highlight the insufficiency of key metabolites as a metabolic barrier for efficient hair cell reprogramming.

Deletion of Luzp2 Does Not Cause Hearing Loss in Mice.

Deafness is the prevailing sensory impairment among humans, impacting every aspect of one's existence. Half of congenital deafness cases are attributed to genetic factors. Studies have shown that Luzp2 is expressed in hair cells (HCs) and supporting cells of the inner ear, but its specific role in hearing remains unclear. To determine the importance of Luzp2 in auditory function, we generated mice deficient in Luzp2. Our results revealed that Luzp2 has predominant expression within the HCs and pillar cells. However, the loss of Luzp2 did not result in any changes in auditory threshold. HCs or synapse number and HC stereocilia morphology in Luzp2 knockout mice did not show any notable distinctions. This was the first study of the role of Luzp2 in hearing in mice, and our results provide important guidance for the screening of deafness genes.

Dock4 is required for the maintenance of cochlear hair cells and hearing function.

Auditory hair cells (HCs) are the mechanosensory receptors of the cochlea, and HC loss or malfunction can result from genetic defects. Dock4, a member of the Dock180-related protein superfamily, is a guanine nucleotide exchange factor for Rac1, and previous reports have shown that Dock4 mutations are associated with autism spectrum disorder, myelodysplastic syndromes, and tumorigenesis. Here, we found that Dock4 is highly expressed in the cochlear HCs of mice. However, the role of Dock4 in the inner ear has not yet been investigated. Taking advantage of the piggyBac transposon system, Dock4 knockdown (KD) mice were established to explore the role of Dock4 in the cochlea. Compared to wild-type controls, Dock4 KD mice showed significant hearing impairment from postnatal day 60. Dock4 KD mice showed hair bundle deficits and increased oxidative stress, which eventually led to HC apoptosis, late-onset HC loss, and progressive hearing loss. Furthermore, molecular mechanism studies showed that Rac1/ß-catenin signaling was significantly downregulated in Dock4 KD cochleae and that this was the cause for the disorganized stereocilia and increased oxidative stress in HCs. Overall, our work demonstrates that the Dock4/Rac1/ß-catenin signaling pathway plays a critical role in the maintenance of auditory HCs and hearing function.

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.

Two entry tunnels in mouse TAAR9 suggest the possibility of multi-entry tunnels in olfactory receptors.

Orthosteric binding sites of olfactory receptors have been well understood for ligand-receptor interactions. However, a lack of explanation for subtle differences in ligand profile of olfactory receptors even with similar orthosteric binding sites promotes more exploration into the entry tunnels of the receptors. An important question regarding entry tunnels is the number of entry tunnels, which was previously believed to be one. Here, we used TAAR9 that recognizes important biogenic amines such as cadaverine, spermine, and spermidine as a model for entry tunnel study. We identified two entry tunnels in TAAR9 and described the residues that form the tunnels. In addition, we found two vestibular binding pockets, each located in one tunnel. We further confirmed the function of two tunnels through site-directed mutagenesis. Our study challenged the existing views regarding the number of entry tunnels in the subfamily of olfactory receptors and demonstrated the possible mechanism how the entry tunnels function in odorant recognition.

Delayed postoperative complications in 624 consecutive cochlear implantation cases.

Background: Sensorineural hearing loss can be cured by cochlear implantation (CI), but complications can occur. Based on when the complications develop, they are categorized as intraoperative complications, early postoperative complications, or delayed postoperative complications (>3 months after the surgery).Aims/objectives: We aimed to investigate the occurrence of delayed complications after CI surgery, and identify appropriate management methods.Material and methods: We analyzed 624 sensorineural hearing loss patients who had been consecutively treated with CI using the conventional surgical technique in our institution and had been followed-up until September 2017.Results: A total of 43 (6.86%) patients out of the 624 CIs (627 ears) reported complications, and 9 (1.44%) were major complications and 34 (5.42%) were minor complications. Wound infection and device failure were the most common major complications, and hematoma was the most common minor complication.Conclusions and significance: CI surgery is a relatively mature technology; the incidence of complications is low, and with early diagnosis and treatment most complications have a good prognosis. Head trauma was the main reason for children's complications, and patients and guardians should be given good education preoperatively about how to manage the CI postoperatively.

Disruption of the autism-related gene Pak1 causes stereocilia disorganization, hair cell loss, and deafness in mice.

Several clinical studies have reported that hearing loss is correlated with autism in children. However, little is known about the underlying mechanism between hearing loss and autism. p21-activated kinases (PAKs) are a family of serine/threonine kinases that can be activated by multiple signaling molecules, particularly the Rho family of small GTPases. Previous studies have shown that Pak1 mutations are associated with autism. In the present study, we take advantage of Pak1 knockout (Pak1-/-) mice to investigate the role of PAK1 in hearing function. We find that PAK1 is highly expressed in the postnatal mouse cochlea and that PAK1 deficiency leads to hair cell (HC) apoptosis and severe hearing loss. Further investigation indicates that PAK1 deficiency downregulates the phosphorylation of cofilin and ezrin-radixin-moesin and the expression of ßII-spectrin, which further decreases the HC synapse density in the basal turn of cochlea and disorganized the HC stereocilia in all three turns of cochlea in Pak1-/- mice. Overall, our work demonstrates that the autism-related gene Pak1 plays a crucial role in hearing function. As the first candidate gene linking autism and hearing loss, Pak1 may serve as a potential target for the clinical diagnosis of autism-related hearing loss.

Clinical application of collagen membrane with umbilical cord-derived mesenchymal stem cells to repair nasal septal perforation.

Objective. We aimed to investigate the clinical efficacy of collagen membrane with umbilical cord-derived mesenchymal stem cells in the endoscopic repair of nasal septal perforation.Methods.We performed a prospective clinical trial between March 2017 and October 2019. Nasal septal perforations were repaired by the endoscopic sandwich technique with the collagen membrane and umbilical cord-derived mesenchymal stem cells. These patients were followed up postoperatively. Their outcomes were comprehensively evaluated by assessing the healing process of the perforations, the visual analog scale (VAS) for nasal discomfort, and the nasal mucociliary transit time (MTT) for the regenerated nasal mucosa.Results. Our study included a total of eight patients with nasal septal perforation (six males and two females, age 36.6 ± 12.8 years, diameter of perforation 1.0 ± 0.2 cm). Seven patients successfully underwent surgical repair. These patients had significantly improved VAS scores 1 month after the operations (1.1 ± 0.4) compared with the preoperative period (5.9 ± 0.7) (P< 0.05). Although the nasal MTT in the nasal septum and the inferior turbinate surface were within the normal limits before the operation and at 1 month after the operation, the postoperative transit time (11.1 ± 2.0 m) was significantly shorter than the preoperative transit time (12.1 ± 2.4 m) (P< 0.05). There were no recurrences of perforation, scab formations, or epistaxis after the operation.Conclusions. The application of the collagen membrane with umbilical cord-derived mesenchymal stem cells is a simple and feasible endoscopic procedure to repair perforated nasal septa and restore satisfactory functional mucosa.

Loss of ARHGEF6 Causes Hair Cell Stereocilia Deficits and Hearing Loss in Mice.

ARHGEF6 belongs to the family of guanine nucleotide exchange factors (GEFs) for Rho GTPases, and it specifically activates Rho GTPases CDC42 and RAC1. Arhgef6 is the X-linked intellectual disability gene also known as XLID46, and clinical features of patients carrying Arhgef6 mutations include intellectual disability and, in some cases, sensorineural hearing loss. Rho GTPases act as molecular switches in many cellular processes. Their activities are regulated by binding or hydrolysis of GTP, which is facilitated by GEFs and GTPase-activating proteins, respectively. RAC1 and CDC42 have been shown to play important roles in hair cell (HC) stereocilia development. However, the role of ARHGEF6 in inner ear development and hearing function has not yet been investigated. Here, we found that ARHGEF6 is expressed in mouse cochlear HCs, including the HC stereocilia. We established Arhgef6 knockdown mice using the clustered regularly interspaced short palindromic repeat-associated Cas9 nuclease (CRISPR-Cas9) genome editing technique. We showed that ARHGEF6 was indispensable for the maintenance of outer hair cell (OHC) stereocilia, and loss of ARHGEF6 in mice caused HC stereocilia deficits that eventually led to progressive HC loss and hearing loss. However, the loss of ARHGEF6 did not affect the synapse density and did not affect the mechanoelectrical transduction currents in OHCs at postnatal day 3. At the molecular level, the levels of active CDC42 and RAC1 were dramatically decreased in the Arhgef6 knockdown mice, suggesting that ARHGEF6 regulates stereocilia maintenance through RAC1/CDC42.

Effects of glucocorticoid on the expression and regulation of aquaporin 5 in the paranasal sinus of rats with chronic rhinosinusitis.

Aquaporins (AQPs) are water-specific membrane channel proteins that regulate water homeostasis for cells and organisms. AQP5 serves an important role in the maintenance of mucosal water homeostasis, and potentially contributes to mucosal edema and inflammation formation in chronic rhinosinusitis (CRS). The aim of the present study was to explore the expression pattern of AQP5 and the effect of glucocorticoids on AQP5 expression in rats with CRS. The rats were randomly divided into three equal groups, as follows: CRS, dexamethasone (dexa) treatment and control groups. A polyvinyl acetal material containing Staphylococcus aureus was inserted into the left nasal cavity of each rat from the CRS and dexa groups. On the 90th post-operative day, the dexa group received dexamethasone (2 mg/kg/day) via intraperitoneal injection for 7 days. The controls did not receive any treatment. The expression of AQP5 in the sinonasal mucosa was determined using immunohistochemistry and quantitative PCR. The immunoreactivities of AQP5 were primarily noted in the epithelial lining and glandular cells, the vascular endothelium and in the goblet cells in the sinonasal mucosa. The AQP5 mRNA expression level was significantly higher in the dexa group than in the control and CRS groups (P=0.006 and P=0.014, respectively). However, no significant difference was indicated between the CRS and control groups (P=0.760). In conclusion, the current study suggests that glucocorticoids induce AQP5 expression in the sinonasal mucosa of CRS rats, which highlights AQP5 as a potential target in the diagnosis and treatment of CRS.

Characterization of the Transcriptomes of Lgr5+ Hair Cell Progenitors and Lgr5- Supporting Cells in the Mouse Cochlea.

Cochlear supporting cells (SCs) have been shown to be a promising resource for hair cell (HC) regeneration in the neonatal mouse cochlea. Previous studies have reported that Lgr5+ SCs can regenerate HCs both in vitro and in vivo and thus are considered to be inner ear progenitor cells. Lgr5+ progenitors are able to regenerate more HCs than Lgr5- SCs, and it is important to understand the mechanism behind the proliferation and HC regeneration of these progenitors. Here, we isolated Lgr5+ progenitors and Lgr5- SCs from Lgr5-EGFP-CreERT2/Sox2-CreERT2/Rosa26-tdTomato mice via flow cytometry. As expected, we found that Lgr5+ progenitors had significantly higher proliferation and HC regeneration ability than Lgr5- SCs. Next, we performed RNA-Seq to determine the gene expression profiles of Lgr5+ progenitors and Lgr5- SCs. We analyzed the genes that were enriched and differentially expressed in Lgr5+ progenitors and Lgr5- SCs, and we found 8 cell cycle genes, 9 transcription factors, and 24 cell signaling pathway genes that were uniquely expressed in one population but not the other. Last, we made a protein-protein interaction network to further analyze the role of these differentially expressed genes. In conclusion, we present a set of genes that might regulate the proliferation and HC regeneration ability of Lgr5+ progenitors, and these might serve as potential new therapeutic targets for HC regeneration.

D-4F, an apolipoprotein A-I mimetic, inhibits TGF-ß1 induced epithelial-mesenchymal transition in human alveolar epithelial cell.

Emerging evidences support that transforming growth factor ß1 (TGF-ß1) induced epithelial-mesenchymal transition (EMT) participates in the pathogenesis of pulmonary fibrosis and asthmatic airway remodeling. Recent studies demonstrated that apolipoprotein A-I (Apo A-I) is the only known substance that can resolve established pulmonary fibrotic nodules, and Apo A-I mimetic D-4F (a synthetic polypeptide consisting of 18 amino acids) plays an inhibitory role in murine asthmatic model. However, cellular mechanisms for such therapeutic effects of Apo A-I and D-4F remain to be elucidated. This study evaluated the effects of D-4F on TGF-ß1 induced EMT in human type II alveolar epithelial cell line A549. A549 cells treated with 10ng/ml of TGF-ß1 manifested distinct EMT, including fibroblastic morphological changes, down-regulation of epithelial marker E-cadherin and up-regulation of mesenchymal marker vimentin. These EMT related changes were all inhibited by D-4F in a concentration dependent manner. Transcriptional investigation demonstrated clearly that D-4F dose-dependently compensated for the reduced E-cadherin mRNA level and the increased vimentin mRNA level in TGF-ß1 treated A549 cells. Translational analysis revealed that D-4F significantly reversed the TGF-ß1 induced changes of E-cadherin and vimentin levels. These results suggested that D-4F inhibits TGF-ß1 induced EMT in human alveolar epithelial cell. Given the functional similarities between D-4F and Apo A-I, it is speculated that D-4F and Apo A-I are able to exert possible anti-fibrotic and anti-asthmatic effects via inhibiting alveolar EMT, and D-4F may possess beneficial clinical potential for patients suffering from pulmonary fibrosis and asthma.