Dock4在小鼠内耳发育与功能中的作用及其分子机制研究

The study found that more than 50% of the sensorineural hearing loss was caused by genetic factors. Thus, it is of great clinical significance to deeply understand the genetic causes of deafness. Dock4 involves in many cell-life activities by activating small G proteins as an atypical guanine nucleotide exchange factors (GEFs) for Dock-B subfamily. A yeast two-hybrid assay showed that Dock4 directly interacts with Harmonin in the inner ear, but there is no systematic study of the role of Dock4 in inner ear development and function. Currently, we have generated mice that Dock4 was deleted. Previous data showed that the hair cell of P60 Dock4 knockout mice appeared dot-like deletion and ciliary polarity change, and accompanied by hearing loss. However, the mechanism of Dock4 in hair cell function remains unclear. This project was, therefore, devised to systematically explore the effect of Dock4 gene on the development, function and survival of hair cells as well as its molecular mechanism using the Dock4 knockout mice. The objectives of the project are: 1. to study the expression and localization of Dock4 in the mouse cochlear at different time points; 2. to study the effect of Dock4 on the development, function and survival of hair cell at different time points and its influence on auditory function; 3. to study the specific molecular mechanism of Dock4 in regulating hair cell development, function and survival. The goal of this project is to establish the experimental and theoretical basis for the clinical prevention and treatment of genetic deafness caused by related genes.

50%以上感音神经性聋是由遗传因素引起的，了解耳聋的遗传因素及其致病机制具有显著意义。Dock4是非典型鸟苷酸交换因子（GEFs）Dock-B亚家族成员，通过激活小G蛋白参与多种细胞生命活动。相关实验表明，Dock4在内耳静纤毛中与Harmonin相互作用，然而Dock4在内耳发育与功能中的作用尚未系统研究。本项目已构建Dock4敲除小鼠，前期数据表明P60的Dock4敲除小鼠毛细胞缺失、纤毛极性改变，并伴随听力下降。然而，Dock4对毛细胞发育功能的影响及其作用机制还不清楚。本项目将通过Dock4敲除小鼠研究Dock4在毛细胞发育功能存活中的作用及分子机制，拟实现以下目标：一、研究Dock4在小鼠耳蜗发育中的时空表达；二、研究Dock4对毛细胞发育功能存活的影响及对听觉的影响；三、研究Dock4调控毛细胞发育功能存活的的分子机制。本项目将为防治相关基因导致的遗传性聋提供实验和理论基础。

听觉毛细胞是耳蜗的机械感觉感受器，毛细胞的丢失或功能障碍可由遗传的缺陷引起。Dock4是Dock180相关蛋白超家族的一员，是Rac1的鸟嘌呤核苷酸交换因子，先前的报道表明，Dock4突变与自闭症谱系障碍、骨髓增生异常综合征和肿瘤发生有关。在这里，我们发现Dock4在小鼠耳蜗hc中高表达。然而，Dock4在内耳中的作用尚未被研究。利用piggyBac转座子系统，建立Dock4敲低小鼠，探讨Dock4在耳蜗中的作用。与野生型对照组相比，Dock4 敲低小鼠在出生后第60天开始就表现出明显的听力障碍。Dock4 敲低小鼠表现出毛束缺陷和氧化应激增加，最终导致毛细胞细胞凋亡、迟发性毛细胞损失和进行性听力损失。此外，分子机制研究表明，Dock4 敲低的耳蜗中Rac1/-catenin信号通路显著下调，这是导致静纤毛紊乱和氧化应激增加的原因。