ABCC1 deficiency potentiated noise-induced hearing loss in mice by impairing cochlear antioxidant capacity.

The ABCC1 gene belongs to the ATP-binding cassette membrane transporter superfamily, which plays a crucial role in the efflux of various endogenous and exogenous substances. Mutations in ABCC1 can result in autosomal dominant hearing loss. However, the specific roles of ABCC1 in auditory function are not fully understood. Through immunofluorescence, we found that ABCC1 was expressed in microvascular endothelial cells (ECs) of the stria vascularis (StV) in the murine cochlea. Then, an Abcc1 knockout mouse model was established by using CRISPR/Cas9 technology to elucidate the role of ABCC1 in the inner ear. The ABR threshold did not significantly differ between WT and Abcc1-/- mice at any age studied. After noise exposure, the ABR thresholds of the WT and Abcc1-/- mice were significantly elevated. Interestingly, after 14 days of noise exposure, ABR thresholds largely returned to pre-exposure levels in WT mice but not in Abcc1-/- mice. Our subsequent experiments showed that microvascular integrity in the StV was compromised and that the number of outer hair cells and the number of ribbons were significantly decreased in the cochleae of Abcc1-/- mice post-exposure. Besides, the production of ROS and the accumulation of 4-HNE significantly increased. Furthermore, StV microvascular ECs were cultured to elucidate the role of ABCC1 in these cells under glucose oxidase challenge. Notably, 30 U/L glucose oxidase (GO) induced severe oxidative stress damage in Abcc1-/- cells. Compared with WT cells, the ROS and 4-HNE levels and the apoptotic rate were significantly elevated in Abcc1-/- cells. In addition, the reduced GSH/GSSG ratio was significantly decreased in Abcc1-/- cells after GO treatment. Taken together, Abcc1-/- mice are more susceptible to noise-induced hearing loss, possibly because ABCC1 knockdown compromises the GSH antioxidant system of StV ECs. The exogenous antioxidant N-acetylcysteine (NAC) may protect against oxidative damage in Abcc1-/- murine cochleae and ECs.

Construction and validation of a folate metabolism-related gene signature for predicting prognosis in HNSCC.

PURPOSE: Metabolic reprogramming is currently considered a hallmark of tumor and immune development. It is obviously of interest to identify metabolic enzymes that are associated with clinical prognosis in head and neck squamous cell carcinomas (HNSCC). METHODS: Candidate genes were screened to construct folate metabolism scores by Cox regression analysis. Functional enrichment between high- and low-folate metabolism groups was explored by GO, KEGG, GSVA, and ssGSEA. EPIC, MCPcounter, and xCell were utilized to explore immune cell infiltration between high- and low-folate metabolism groups. Relevant metabolic scores were calculated and visually analyzed by the "IOBR" software package. RESULTS: To investigate the mechanism behind metabolic reprogramming of HNSCC, 2886 human genes associated with 86 metabolic pathways were selected. Folate metabolism is significantly enriched in HNSCC, and that the six-gene (MTHFD1L, MTHFD2, SHMT2, ATIC, MTFMT, and MTHFS) folate score accurately predicts and differentiates folate metabolism levels. Reprogramming of folate metabolism affects CD8T cell infiltration and induces immune escape through the MIF signaling pathway. Further research found that SHMT2, an enzyme involved in folate metabolism, inhibits CD8T cell infiltration and induces immune escape by regulating the MIF/CD44 signaling axis, which in turn promotes HNSCC progression. CONCLUSIONS: Our study identified a novel and robust folate metabolic signature. A folate metabolic signature comprising six genes was effective in assessing the prognosis and reflecting the immune status of HNSCC patients. The target molecule of folate metabolic reprogramming, SHMT2, probably plays a very important role in HNSCC development and immune escape.

Susceptibility of immature spiral ganglion neurons to aminoglycoside-induced ototoxicity is mediated by the TRPV1 channel in mice.

Aminoglycoside antibiotics are among the most common agents that can cause sensorineural hearing loss. From clinical experience, premature babies, whose inner ear is still developing, are more susceptible to aminoglycoside-induced ototoxicity, which is echoed by our previous study carried out in organotypic cultures. This study aimed to investigate whether a nonselective cation channel, TRPV1, contributes to the susceptibility of immature spiral ganglion neurons (SGNs) to the damage caused by aminoglycosides. Through western blotting and immunofluorescence, we found that the TRPV1 expression levels were much higher in immature SGNs than in their mature counterparts. In postnatal day 7 cochlear organotypic cultures, AMG-517 reduced reactive oxygen species generation and inhibited SGN apoptosis under aminoglycoside challenge. However, in adult mice, AMG-517 did not ameliorate the ABR threshold increase at high frequencies (16 kHz and 32 kHz) after aminoglycoside administration, and the SGNs within the cochleae had no morphological changes. By further regulating the function of TRPV1 in primary cultured SGNs with its inhibitor AMG-517 and agonist capsaicin, we demonstrated that TRPV1 is a major channel for aminoglycoside uptake: AMG-517 can significantly reduce, while capsaicin can significantly increase, the uptake of GTTR. In addition, TRPV1 knockdown in SGNs can also significantly reduce the uptake of GTTR. Taken together, our results demonstrated that aminoglycosides can directly enter immature SGNs through the TRPV1 channel. High expression of TRPV1 contributes to the susceptibility of immature SGNs to aminoglycoside-induced damage. The TRPV1 inhibitor AMG-517 has the potential to be a therapeutic agent for preventing aminoglycoside-induced ototoxicity in immature SGNs.

A Novel EYA1 Mutation Causing Alternative RNA Splicing in a Chinese Family With Branchio-Oto Syndrome: Implications for Molecular Diagnosis and Clinical Application.

OBJECTIVES: Branchio-oto syndrome (BOS) primarily manifests as hearing loss, preauricular pits, and branchial defects. EYA1 is the most common pathogenic gene, and splicing mutations account for a substantial proportion of cases. However, few studies have addressed the structural changes in the protein caused by splicing mutations and potential pathogenic factors, and several studies have shown that middle-ear surgery has limited effectiveness in improving hearing in these patients. BOS has also been relatively infrequently reported in the Chinese population. This study explored the genetic etiology in the family of a proband with BOS and provided clinical treatment to improve the patient's hearing. METHODS: We collected detailed clinical features and peripheral blood samples from the patients and unaffected individuals within the family. Pathogenic mutations were identified by whole-exome sequencing and cosegregation analysis and classified according to the American College of Medical Genetics and Genomics guidelines. Alternative splicing was verified through a minigene assay. The predicted three-dimensional protein structure and biochemical experiments were used to investigate the pathogenicity of the mutation. The proband underwent middle-ear surgery and was followed up at 1 month and 6 months postoperatively to monitor auditory improvement. RESULTS: A novel heterozygous EYA1 splicing variant (c.1050+4 A>C) was identified and classified as pathogenic (PVS1(RNA), PM2, PP1). Skipping of exon 11 of the EYA1 pre-mRNA was confirmed using a minigene assay. This mutation may impair EYA1-SIX1 interactions, as shown by an immunoprecipitation assay. The EYA1-Mut protein exhibited cellular mislocalization and decreased protein expression in cytological experiments. Middle-ear surgery significantly improved hearing loss caused by bone-conduction abnormalities in the proband. CONCLUSION: We reported a novel splicing variant of EYA1 in a Chinese family with BOS and revealed the potential molecular pathogenic mechanism. The significant hearing improvement observed in the proband after middle-ear surgery provides a reference for auditory rehabilitation in similar patients.

Gene regulation analysis of patient-derived iPSCs and its CRISPR-corrected control provides a new tool for studying perturbations of ELMOD3 c.512A>G mutation during the development of inherited hearing loss.

The ELMOD3 gene is implicated in causing autosomal recessive/dominant non-syndromic hearing loss in humans. However, the etiology has yet to be completely elucidated. In this study, we generated a patient-derived iPSC line carrying ELMOD3 c.512A>G mutation. In addition, the patient-derived iPSC line was corrected by CRISPR/Cas9 genome editing system. Then we applied RNA sequencing profiling to compare the patient-derived iPSC line with different controls, respectively (the healthy sibling-derived iPSCs and the CRISPR/Cas9 corrected iPSCs). Functional enrichment and PPI network analysis revealed that differentially expressed genes (DEGs) were enriched in the gene ontology, such as sensory epithelial development, intermediate filament cytoskeleton organization, and the regulation of ion transmembrane transport. Our current work provided a new tool for studying how disruption of ELMOD3 mechanistically drives hearing loss.

Genetic research progress in branchio-oto syndrome/ branchio-oto-renal syndrome. / 鳃耳综合征/é³ƒè€³è‚¾ç»¼åˆå¾çš„é—ä¼ å­¦ç ”ç©¶è¿›å±•.

Branchio-oto syndrome (BOS)/branchio-oto-renal syndrome (BORS) is a kind of autosomal dominant heterogeneous disorder. These diseases are mainly characterized by hearing impairment and abnormal phenotype of ears, accompanied by renal malformation and branchial cleft anomalies including cyst or fistula, with an incidence of 1/40 000 in human population. Otic anormalies are one of the most obvious clinical manifestations of BOS/BORS, including deformities of external, middle, inner ears and hearing loss with conductive, sensorineural or mix, ranging from mild to profound loss. Temporal bone imaging could assist in the diagnosis of middle ear and inner ear malformations for clinicians. Multiple methods including direct sequencing combined with next generation sequencing (NGS), multiplex ligation-dependent probe amplification (MLPA), or array-based comparative genomic hybridization (aCGH) can effectively screen and identify pathogenic genes and/or variation types of BOS/BORS. About 40% of patients with BOS/BORS carry aberrations of EYA1 gene which is the most important cause of BOS/BORS. A total of 240 kinds of pathogenic variations of EYA1 have been reported in different populations so far, including frameshift, nonsense, missense, aberrant splicing, deletion and complex rearrangements. Human Endogenous Retroviral sequences (HERVs) may play an important role in mediating EYA1 chromosomal fragment deletion mutations caused by non-allelic homologous recombination. EYA1 encodes a phosphatase-transactivator cooperated with transcription factors of SIX1, participates in cranial sensory neurogenesis and development of branchial arch-derived organs, then regulates the morphological and functional differentiation of the outer ear, middle ear and inner ear toward normal tissues. In addition, pathogenic mutations of SIX1 and SIX5 genes can also cause BOS/BORS. Variations of these genes mentioned above may cause disease by destroying the bindings between SIX1-EYA1, SIX5-EYA1 or SIX1-DNA. However, the role of SIX5 gene in the pathogenesis of BORS needs further verification.

Otological manifestations in branchiootorenal spectrum disorder: A systematic review and meta-analysis.

Branchiootorenal spectrum disorder (BORSD) is a group of rare autosomal dominant entities characterized by branchiogenic malformations, hearing loss (HL) and renal anomalies. It comprises branchiootorenal syndrome and branchiootic syndrome, distinguished by the presence or absence of renal abnormalities. Pathogenic variants have been discovered in the following genes: EYA1, SIX5, SIX1 and SALL1. As the otological phenotype in BORSD is inconsistently reported, we performed a systematic review to provide an up-to-date overview, correlated with the genotype. Forty publications were included, describing 295 individual patients. HL was diagnosed in 95%, usually bilateral and mixed-type, and differed among the different genes involved. Mixed moderate-to-severe HL was the predominant finding in patients with EYA1 involvement, regardless of the presence of renal abnormalities. The sensorineural HL of profound severity was more prevalent in patients with SIX1 mutations. No significant differences among different mutation types or location within the genes could be observed. Structural otological manifestations, ranging from periauricular to inner ear anomalies, were common in both genes. Especially periauricular anomalies were more common and more severe in EYA1. In summary, otological differences among the different genes involved in BORSD are observed, so the molecular analysis is strongly advised.

Elmod3 knockout leads to progressive hearing loss and abnormalities in cochlear hair cell stereocilia.

ELMOD3, an ARL2 GTPase-activating protein, is implicated in causing hearing impairment in humans. However, the specific role of ELMOD3 in auditory function is still far from being elucidated. In the present study, we used the CRISPR/Cas9 technology to establish an Elmod3 knockout mice line in the C57BL/6 background (hereinafter referred to as Elmod3-/- mice) and investigated the role of Elmod3 in the cochlea and auditory function. Elmod3-/- mice started to exhibit hearing loss from 2 months of age, and the deafness progressed with aging, while the vestibular function of Elmod3-/- mice was normal. We also observed that Elmod3-/- mice showed thinning and receding hair cells in the organ of Corti and much lower expression of F-actin cytoskeleton in the cochlea compared with wild-type mice. The deafness associated with the mutation may be caused by cochlear hair cells dysfunction, which manifests with shortening and fusion of inner hair cells stereocilia and progressive degeneration of outer hair cells stereocilia. Our finding associates Elmod3 deficiencies with stereocilia dysmorphologies and reveals that they might play roles in the actin cytoskeleton dynamics in cochlear hair cells, and thus relate to hearing impairment.

Novel insights into ion channels in cancer stem cells (Review).

Cancer stem cells (CSCs) are immortal cells in tumor tissues that have been proposed as the driving force of tumorigenesis and tumor invasion. Previously, ion channels were revealed to contribute to cancer cell proliferation, migration and apoptosis. Recent studies have demonstrated that ion channels are present in various CSCs; however, the functions of ion channels and their mechanisms in CSCs remain unknown. The present review aimed to focus on the roles of ion channels in the regulation of CSC behavior and the CSC-like properties of cancer cells. Evaluation of the relationship between ion channels and CSCs is critically important for understanding malignancy.