CNTN4调节GluR1在听神经病谱系障碍中的致病机制研究

Auditory neuropathy spectrum disorder (ANSD) is one of the most harmful diseases leading to human hearing impairment. A large amount of genetic evidence shows that the CNTNs family members of nerve adhesion molecules is closely related to neurodevelopmental diseases, including auditory nerve diseases. In the early stage, we found that the pathogenic cause of a patient with ANSD was CNV in CNTN4. In order to study the relationship between CNTN4 and ANSD, we constructed CNTN4 knockout (KO) mice, and found that KO mice had the phenotype of ANSD, and further found that the gene expressing glutamate receptor GluR1 was significantly reduced in the cochlea. Based on previous studies, we proposed the scientific hypothesis that CNTN4 regulates GluR1 and led to the occurrence of ANSD. This project will study the pathogenic mechanism of CNTN4 in ANSD from the following aspects :(1) to detect the neurons expressing CNTN4 in the auditory nerve afferent pathway and the effect of CNTN4 on neuron development; (2) To investigate the molecular mechanism of CNTN4 that cause spiral ganglion cells damage and occurance of ANSD through regulating GluR1; (3) to study whether the addition of interference agent can save the hearing impairment of KO mice. At the same time we will screen the mutations of CNTN4 in ANSD population. The accomplishment of this study is expected to find a new ANSD pathogenic gene, and provide potential targets and theoretical basis for drug treatment of ANSD.

听神经病谱系障碍(ANSD)是导致人类听觉障碍中危害性最大的疾病之一，大量遗传学证据显示神经粘附分子CNTNs家族成员与神经发育性疾病包括听神经疾病密切相关，前期我们发现一ANSD患者的致病原因为CNTN4基因的拷贝数变异。我们构建了CNTN4基因敲除小鼠，结果发现敲除小鼠具有ANSD的表型，同时发现表达GluR1的基因在其耳蜗中明显降低。基于前期研究，我们提出科学假设：CNTN4调节GluR1导致ANSD的发生。本项目将从三个方面研究CNTN4在ANSD中的具体致病机制：(1)研究CNTN4在蜗神经传入通路上所表达的神经元及对神经元发育的作用；(2)研究CNTN4调节GluR1致螺旋神经元损伤引起ANSD的分子机制；(3)研究加入干扰剂能否挽救敲除小鼠的听觉障碍。本研究的顺利实施有望发现ANSD新的致病基因，并为ANSD的药物治疗提供潜在的靶点及理论依据。

在本项目实施过程中，我们完成了四个方面的工作：(1)收集了200例听神经病谱系障碍(ANSD)散发患者，提取外周血淋巴细胞基因组DNA并进行CNTN4基因突变检测，构建了中国ANSD患者的CNTN4基因突变图谱，并收集患者及家系成员的详细临床表型，进行表型-基因型的相关性分析；(2)利用免疫组化及免疫荧光等方法明确了Cntn4在蜗神经传入通路上所表达的神经元及对神经元发育的作用，我们发现Cntn4在出生后耳蜗螺旋神经元的突触前膜定位最明显，并且随着年龄的增大逐渐减少，到成年后维持稳定。利用原位杂交与免疫荧光方法发现Cntn4与GluR1在蜗神经传入通路上是共定位的，Cntn4基因敲除小鼠耳蜗在形态学上与野生小鼠无明显差别，螺旋神经节细胞及轴突数量减少；(3)利用Western blot检测GluR1在Cntn4基因敲除小鼠脑干听区的蛋白质水平明显降低；并且利用免疫共沉淀技术鉴定了Cntn4与GluR1存在相互作用；(4)利用螺旋神经元计数、TUNEL染色、活化的caspase3检测和流式细胞技术等方法在原代螺旋神经元中检测细胞凋亡情况，发现Cntn4突变小鼠内毛细胞及耳蜗Corti器无明显改变、螺旋神经元轴突及树突出现凋亡。上述研究结果提示我们CNTN4可能是ANSD新的致病基因，同时为ANSD的药物治疗提供潜在的靶点及理论依据。在本项目资助下，项目负责人以一作或通讯作者身份在国际性学术刊物上发表SCI论文6篇，共培养1名博士研究生和2名硕士研究生。

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