遗传性耳聋基因TBC1D24的内耳功能及致聋机制研究

TBC1D24 was recently identified as a deafness gene. Mutations in TBC1D24 may lead to genetic hearing loss with variable phenotypes. The inner ear function and the pathogenic mechanism of TBC1D24 are not clear. In previous studies, we revealed that the p.S178L mutation of TBC1D24 may lead to autosomal dominant non-syndromic deafness with significantly different phenotypes from its recessive counterpart. We showed that in the mouse inner ear, Tbc1d24 is specifically expressed in the spiral ganglion neurons and the stereocilia of the hair cells. Our in vitro study showed that the p.S178L mutation resulted in a significant increase of TBC1D24’s ability to prompt cellular elongation and arborization, suggesting that it is a gain-of-function mutation. In this study, we propose to generate a conditional knock-out mouse model for Tbc1d24. By analyses of this model in auditory physiology, inner ear pathology and molecular biology, we aim to elucidate the function and the underlying pathways of TBC1D24 in different locations of inner ear, and to explore the pathogenic mechanism associated with the loss-of-function mutations of TBC1D24. In addition, we propose to generate a knock-in mouse model for the p.S178L mutation of TBC1D24. By comparative study of the knock-in and the knock-out mouse models, we aim to understand the unique pathogenic mechanism associated with the p.S178L mutation. The proposed study of TBC1D24 may provide important insights into the mechanism of hearing and deafness and may improve the diagnosis, intervention and prevention of the associated hearing impairment.

TBC1D24为一新近发现的耳聋基因，其不同类型突变可导致具有多样化临床表型的遗传性耳聋，但在内耳的具体功能和突变致聋机制不明。申请者课题组在前期研究中发现该基因p.S178L杂合突变可导致显性非综合征性耳聋，听力表型显著有别于隐性突变。我们发现Tbc1d24在小鼠内耳特异性表达于螺旋神经节神经元及毛细胞静纤毛，体外研究显示p.S178L突变导致TBC1D24促细胞树突状分支能力的异常性升高，可能为功能获得性突变。本申请拟构建Tbc1d24条件性敲除小鼠，通过听觉生理、内耳病理及分子生物学等研究手段明确该基因在内耳不同表达区域的具体功能和作用通路，探索其功能缺失性突变的致聋机制；构建p.S178L突变敲入小鼠，通过和Tbc1d24敲除小鼠的比较研究验证该突变所特有的致聋机制。本研究有望以TBC1D24为切入点在分子层面上更深入的理解听觉的发生机制和发病机制，促进相关聋病的诊断、干预和预防。

本项目拟对Tbc1d24基因突变小鼠模型的表型和致聋机制开展深入的功能研究，在分子水平上加深对与TBC1D24基因相关的听觉基础和相关聋病发病机制的理解，为遗传性耳聋的诊断、预防和治疗奠定理论基础。项目按照计划执行，完成情况良好，通过Tbc1d24 基因p.S178L突变小鼠的构建，成功获得了可模拟人类听力表型的动物模型，为后续深入的功能学研究和基因治疗研究提供了重要的研究平台和工具。通过对该突变小鼠的听力学、电生理学、显微及超微结构病理学和分子生物学研究，综合运用脑干诱发电位、膜片钳、免疫荧光耳蜗铺片及切片、扫描及透射电镜、定量蛋白电泳等技术手段，在国际上首次揭示显性遗传性耳聋新致病基因TBC1D24在内耳中的听觉功能与内毛细胞的突触囊泡转运与循环相关。结合离体免疫共沉淀等手段，证实TBC1D24基因p.S178L突变为功能获得性突变，通过抑制拮抗蛋白ARF6的表达及蛋白结合能力，导致TBC1D24功能异常性增高，引起内毛细胞中转运囊泡聚集，干扰突触囊泡的转运和循环，从而引起突变小鼠在长时程高强度刺激下突触囊泡神经递质释放能力的下降，进而引起以ABR阈值上刺激I波波幅下降为代表的隐蔽性听力损失。该突变致聋机制本质上属于听神经突触病变，本研究在听神经突触病变引起隐蔽性听力损失的分子机制研究领域创新性发现了一个新的关键基因，为在分子层面更深入的理解和该类型听力损失密切相关的噪声性聋、老年性聋的发生发展机制提供了新的研究角度和切入点。总体而言本项目完成情况良好，研究成果对相关遗传性耳聋的精准诊断、预防和治疗起到了良好的促进作用。项目目前已发表资金标注的SCI论文6篇，尚有一篇主要研究成果论文在投稿过程中。培养两名博士研究生，以本项目研究为博士论文主要研究内容之一，完成博士研究论文通过答辩并顺利毕业。