Scientific Report: Study on the Mechanism of Deafness in Cx26-Deficient Mice

Introduction

Mutations in the Gjb2 gene are the most common cause of autosomal recessive non-syndromic hereditary deafness, accounting for about 50% of all cases. The Cx26 protein encoded by the Gjb2 gene is mainly expressed in cochlear epithelial supporting cells and is responsible for intercellular communication. For individuals with severe hearing loss caused by Gjb2 gene mutations, cochlear implantation (CI) is the only treatment that can improve hearing. However, the effectiveness of cochlear implants varies, and in addition to clinical factors, the preservation of cochlear neural components is a key factor in achieving good CI results. Therefore, it is crucial to study the pathophysiological changes in the peripheral auditory nervous system of Gjb2 mutant mice.

Research Background and Objectives

Based on the above background, this study aims to explore the cochlear neural system pathological changes caused by Gjb2 gene deletion. The research team constructed a conditional Cx26 knockout mouse model (Cx26-cko) to study the mechanism of changes in this process in detail.

Research Source

This article was written by the following researchers: Yue Qiu, Le Xie, Xiaohui Wang, Kai Xu, Xue Bai, Sen Chen, Yu Sun. They are from the Department of Otolaryngology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, and the Department of Otolaryngology, Nanchang University. The article was accepted for publication in “Neuroscience Bulletin” on September 14, 2023.

Research Design and Methods

Mouse Model Construction

The research team used Cx26loxp/loxp and Sox2-CreER transgenic mice provided by Prof. Lin from Emory University and Prof. Zhang from Southeast University, respectively, to generate Tamoxifen-induced Cx26-cko mice through breeding. The experimental group and control group were Cx26-cko mice and their littermate Cx26loxp/loxp mice, respectively.

Protein Extraction and Western Blot Analysis

On P7 (7 days after birth), Cx26-cko mice and littermate control mice were anesthetized and euthanized. Proteins were extracted from the cochlear membranous labyrinth tissue, separated by electrophoresis, and analyzed by Western Blot to measure the expression level of Cx26 protein.

Auditory Brainstem Response (ABR) and Distortion Product Otoacoustic Emissions (DPOAE) Measurements

At one month of age, mice were anesthetized with a composite anesthetic, and their ABR and DPOAE were measured. In a quiet environment, electrodes were inserted into the external auditory canal to determine the minimum stimulus level that produced repeatable ABR waveforms at various frequencies as the ABR threshold.

Resin Sectioning and Transmission Electron Microscopy (TEM)

The cochlea was fixed, decalcified, embedded, and sectioned. Transmission electron microscopy was used to observe resin sections and ultrathin sections, and quantitative analysis of SGNs was performed.

Real-time Quantitative PCR (RT-qPCR)

RT-qPCR was used to measure the mRNA expression levels of specific genes (such as BDNF, NT3, and their receptors TRKB and TRKC) in the cochlea of mice at AP30.

Research Results

Decreased Cx26 Protein Levels

Immunofluorescence and Western Blot analysis showed that Cx26 protein levels were significantly reduced in the cochlear epithelium of Cx26-cko mice. There was no significant difference in Cx26 protein expression in the cochlear lateral wall and spiral limbus between the control group and the Cx26-cko group.

Severe Hearing Impairment

ABR measurements showed that Cx26-cko mice had severe hearing impairment across the entire frequency range, with significantly elevated ABR thresholds at all frequencies. DPOAE was almost at noise level at most stimulus levels.

Degenerative Changes in Hair Cells and Deiter’s Cells

The number of inner and outer hair cells (IHC and OHC) in Cx26-cko mice was significantly reduced, especially in the middle and basal turns, showing a large loss of Hair cells (Hcs) and Deiter’s cells (Dcs) and disordered cell arrangement.

Type II Spiral Ganglion Neurons (SGNs) Abnormalities

During the overall development of the cochlea, Cx26 deficiency caused abnormalities in type II SGNs, characterized by a reduction in fiber numbers, particularly significant at P11 and P30.

Type I SGNs Abnormalities

Cx26-cko mice showed a significant reduction in type I SGNs nerve endings, leading to the disappearance or significant shrinkage of auditory nerve fiber (ANF) terminals, thereby affecting the conversion of sound signals to the central nervous system.

Demyelination Changes

During cochlear maturation, Cx26 deficiency led to a significant decrease in spiral ganglion neuron density and significant demyelination of nerve cells, greatly affecting neural signal transmission.

Research Conclusions

Through this study, we successfully constructed a Sox2 promoter-driven Cx26 knockout mouse model, revealing a series of pathological changes caused by Cx26 deficiency, including loss of hair cells and Deiter’s cells, abnormal peripheral auditory nerve fibers, and demyelination. These findings provide important insights into understanding the mechanisms of deafness and potential directions for treating severe deafness caused by Cx26 deficiency.

Research Significance

The critical role of Cx26 protein in cochlear supporting cells revealed by this study provides new understanding of cochlear development and auditory signal transmission, emphasizing the importance of maintaining cochlear neural components for successful cochlear implantation. This discovery will aid future research on deafness mechanisms and may promote the development of new treatment strategies.

We look forward to further research revealing the molecular mechanisms of SGN degeneration caused by Cx26 deficiency and exploring new therapeutic approaches to alleviate deafness caused by Gjb2 mutations.