Fascin-2基因TALEN敲除小鼠渐进性听力减退的机制研究

Fascin-2(Fscn2) is an actin cross-linker protein. Mutation in fscn2 gene is related to hearing loss in DBA/2J mice. To fully understand the function of fscn2 gene, we established a mouse model (fscn2-/-) of age-related hearing loss on the B6 background by TALEN technique. We hypothesize that abnormal in structures of filament bundles in hair cells and in transportation of Fscn2-TALEN protein fragment in endoplasmic reticulum is responsible for the hearing loss. Therefore, in this study, we plan, firstly, to identify the features of ultrastructures of the actin filaments by scanning electron microscopy (SEM). Secondly, we will express the Fscn2 and Fscn2-TALEN fragment in vitro, and test their actin binding and bounding ability. We will also localize the positions of the actin isorforms in the filament by immunofluorescent microscopy. Thirdly, we will express the Flag-labeled Fscn2, Fscn2（R109H）and Fscn2-TALEN fragment in HEI-OC1 cells, observe the aggregation of Fscn2-TALEN fragment in endoplasmic reticulum, detect the markers of endoplasmic reticulum stress (ERS) and determine the activation of apoptosis related pathways trigged by ERS. The markers of ERS and apoptosis will be confirmed in the fscn2 mutant mice. The results in this project will provide new ways or targets for prevention and treatment of age-related hearing loss in human.

Fascin-2（Fscn2）是肌动蛋白交联蛋白，fscn2基因突变（R109H）与DBA小鼠的听力减退有关。为深入研究该基因的功能，我们用TALEN 技术敲除B6小鼠的fscn2 基因，小鼠呈早发性渐进性的听力减退。预测Fscn2-TALEN蛋白片段交联肌动蛋白的功能缺失所致的纤毛结构损害及其在内质网的转运障碍引起的毛细胞凋亡在小鼠的听力减退中起重要作用。拟动态电镜观察fscn2敲除小鼠毛细胞纤毛束的超微结构；体外表达Fscn2蛋白和Fscn2-TALEN片段，速度共沉淀确定其与肌动蛋白的结合与交联作用，免疫荧光共定位分析肌动蛋白在fscn2敲除小鼠静纤毛分布的变化；在耳蜗HEI-OC1细胞表达Flag 标记的Fscn2、Fscn2 R109H和Fscn2-TELEN片段，观察变异蛋白的异常转运、内质网应激和特定凋亡通路的活化，在体验证相关通路并干预。为增龄性聋的防治提供新途径或新靶点。

Fascin2（Fscn2）是肌动蛋白交联蛋白，参与毛细胞静纤毛和视网膜结构的构成。Fscn2基因突变可致人和小鼠视网膜变性或小鼠听力损害。为深入研究Fscn2基因的功能，我们采用TALEN技术敲除了C57BL/6J小鼠的Fscn2 基因，纯合子小鼠（Fscn2-/-）呈早发性（出生后21天）渐进性听力减退。本项目旨在研究Fscn2-/-小鼠听力减退的机制。我们发现(1)Fscn2-/-小鼠呈渐进性的耳蜗细胞损害。从3周龄到52周龄，Fscn2-/-小鼠耳蜗底回毛细胞渐进性的缺失，40周龄后，外毛细胞几乎全部缺失。 4周龄至40周龄期间，Fscn2-/-小鼠耳蜗底回SGNs平均细胞密度逐渐降低； 8周龄及以后，Fscn2-/-小鼠耳蜗底回SGNs密度显著低于Fscn2+/+小鼠。(2)Fscn2-/-小鼠内耳PARVB-ILK-AKT相关信号转导通路失调。选取8周龄的Fscn2+/+和Fscn2-/-小鼠做内耳基因表达谱芯片分析，结果显示细胞黏着斑相关分子PARVB在Fscn2-/-小鼠内耳表达显著升高。Western blot 结果显示，4周龄及8周龄Fscn2-/-小鼠PARVB表达上调，ILK和pAKT水平降低，抗凋亡蛋白Bcl-2表达降低，活化Caspase9水平升高；免疫荧光组织化学分析显示，PARVB在Fscn2-/-小鼠耳蜗螺旋神经元的染色强度明显高于Fscn2+/+小鼠，pAKT在Fscn2-/-小鼠螺旋神经元的表达显著低于Fscn2+/+小鼠。(3)FSCN2通过PARVB-ILK-AKT信号转导通路调节耳蜗细胞的增殖、凋亡及迁移。敲低HEI-OC1的Fscn2基因， PARVB的表达水平增高，ILK和pAKT水平降低，抗凋亡蛋白Bcl-2表达降低，活化的Caspase9水平升高, 细胞增殖率降低，迁移速率下降; 而在HEI-OC1细胞过表达Fscn2基因，PARVB表达降低，ILK-pAKT-Bcl2升高。(4)研究还发现Fscn2缺失导致顺铂的耳毒性增强。体内体外实验表明，Fscn2 能缓解顺铂引起的小鼠听力损害和耳蜗细胞凋亡。因此，Fscn2基因敲除小鼠听力减退的主要原因是耳蜗毛细胞和螺旋神经元的渐进变性，FSCN2通过调节PARVB-ILK-AKT相关信号转导通路参与了耳蜗细胞的损害。本研究将为遗传性聋的防治提供新靶点或新思路。