DFNB12新模型小鼠(erl)耳聋的分子机理研究

CDH23 and PCDH15 are the two interactomes of tip links, which regulate the mechanotransduction(MET) channels of hair cells in the organ of Corti in the cochlea. It was reported that the penetrance of human DFNB12 (nonsyndromic deafness) caused by missense mutations in CDH23 was much higher than that of USH1D, which may result from nonsense, splice-site, frame shift or missense mutations in Cdh23. However, interactions of CDH23 with PCDH15 in DFNB12 are not fully understood. In the past few years, we developed a novel mouse model (erl, er long, in Chinese) for DFNB12, which bears a mutation (208 T>C) in Cdh23 gene, causing an amino-acid substitution (S70P)in CDH23. The erl mice were characterized by progressive hearing loss beginning at postnatal day 27 (P27)and being absolutly deafness at P90. Coordinatly, out hair cell(OHC) loss in the erl mice became progressively worse over the time and moved from the cochlear base to the apex. The progressive loss of hearing and OHCs in the erl mice provides a period for mechanistic study and drug intervention to preserve hearing. We hypothesize that alteration in the structures and adhering properties of CDH23 may lead to disruption of tip links, which is responsible for the erl's hearing loss. In this project, therefore, we will prepare the recombinant ectodomains (ECs) of CDH23(S70P) and PCDH15, and observe the interaction of CDH23(S70P) with PCDH15 and the affinity of CDH23(S70P) with Ca+2. We will also have a time-course observation of the features of tip links and the preserving effects on tip links by anti-apoptostic procedures, in the erl mice. The results from this study may aid in providing a new theory on the development of DFNB12 or finding new ways to prevent and treat the diseases.

CDH23和PCDH15的钙离子依赖性相互作用形成了毛细胞的顶连接, 调控耳蜗Corti 器的MET通道。在人类CDH23突变所致的听力障碍中，以错义突变所引起的DFNB12最为常见,其发病机理尚不清楚。我们建立了独特的DFNB12小鼠（erl）模型，基因突变引起CDH23氨基酸的替换(S70P)，导致CDH23二级结构的改变,小鼠表现为出生后27到90天渐进性的听力减退，期间毛细胞缺失从耳蜗底回向顶回蔓延，这种演变过程为erl小鼠耳聋的机理研究和干预提供了良机。我们认为erl突变所导致的毛细胞顶连接的结构与功能的改变是小鼠听力减退的主要原因。本课题拟体外表达erl小鼠的CDH23细胞外结构域，研究CDH23(S70P)与PCDH15的相互作用及与钙离子的亲和力；体内观察erl小鼠毛细胞顶连接的特征及抗凋亡对顶连接的保护作用，为揭示人类DFNB12的发病机理提供新理论或对其防治提供新途径。

erl 小鼠是我们报道的一种新的DFNB12或渐进性聋小鼠模型（Han FC, et al. Pharmacogenomics J, 2012), 其在Cdh23基因上有两个突变位座，分别为ah l(G753A，引起第7个外显子缺失) 和 erl(T208C,引起氨基酸替换S70P), 导致CDH23蛋白第1-2细胞外结构域（ECs）变异。erl小鼠表现为早发性渐进性听力减退，期间毛细胞缺失从耳蜗底回逐渐向顶回蔓延。erl 小鼠为增龄性聋的机理研究和听力恢复性试验提供了时间窗口。本项目执行的主要内容、重要结果和意义如下：. 1. erl 小鼠变异蛋白CDH23-EC1+2 结合Ca2+活性的研究：克隆erl小鼠变异蛋白（CDH23-EC1+2）的基因序列，在大肠杆菌中表达重组蛋白。表达产物主要以包涵体的形式存在，通过8 M 尿素变性和复性，用离子交换和凝胶过滤法纯化重组蛋白。与重组的野生蛋白比较，变异蛋白的钙离子结合能力下降，Ca2+依赖性胰酶降解保护作用显著性降低，这将影响CDH23与PCDH15 的Ca2+ 依赖性相互作用和顶连接（tip-link）的功能，是erl小鼠听力下降的主要机制。. 2. 促红细胞生成素（EPO）对erl小鼠听力的保护研究：我们发现细胞凋亡是erl小鼠听力减退和毛细胞缺失的主要原因，而EPO具有抗细胞凋亡的作用。给出生后7天（P7）的小鼠腹腔内注射EPO，隔天一次。结果显示，与PBS治疗组或非治疗组小鼠比较， EPO能显著降低小鼠的ABR阈值，提升DPOAE幅值，减少耳蜗中回和底回外毛细胞的缺失率（P < 0.05）。这是EPO对渐进性听力减退模型小鼠具有听力保护作用的首次报道，有潜在的应用价值。. 3. Cdh23基因突变(ahl）小鼠（erl、 A/J 和 DBA/2J）的耳聋特点和耳聋机制的研究：erl、 A/J和DBA/2J等小鼠均携带一个Cdh23基因突变位座(ahl），出生后3-4周开始发生渐进性听力减退，毛细胞和螺旋神经元等呈渐进性的变性或缺失，细胞凋亡相关分子caspases 等参与了耳蜗细胞的损害，Z-VAD-FMK或NGF或α-硫辛酸可有效降低Cdh23基因突变小鼠的听力阈值，保护小鼠毛细胞的纤毛结构和减轻螺旋神经元的变性。. 本研究的结果对认识增龄性聋发生机制和对其防治措施的制定均有重要意义。