柠檬酸合成酶基因突变致A/J小鼠听力减退的机制研究

A/J mice are a typical mouse model of age related hearing loss（AHL）and a mutation in gene of citrate synthase (Cs) is related to the hearing loss by a recent study. Our preliminary research showed that high mRNA levels of Caspases occurred in the inner ears of A/J mice at birth day and a pan-caspase inhibitor preserved the hearing of A/J mice. Further study revealed that knockdown of Cs by RNA interference (RNAi) up-regulated caspase-3 in HEI-OC1cells. CS is a key enzyme in tricarboxylic acid cycle. We propose that mutation in Cs gene may lead to endoplasmic reticulum stress (ERS), mitochondrial metabolic disorder and, finally, cell apoptosis, which plays an important role in the hearing loss of A/J mice. Therefore, in the present study, the mutant genes will firstly be cloned and expressed in HEI-OC1 cells. The mutant proteins will be localized and ERS will be identified in the cells. Secondly, cell models with low expression of Cs gene will be developed by RNAi techniques and indicators related to energy metabolic disorders and oxidative stress in the mitochondrion will be detected. Thirdly, key molecules in the apoptotic pathways related to ERS and mitochondrion will be screened and their regulation mechanism be identified. Finally, alteration of key molecules in oxidative stress or apoptotic pathways will be proved in A/J mice and be targeted by specific drugs to improve the mouse hearing. This study aims to explore the molecular mechanism for mutation in Cs gene contributing to hearing loss in A/J mice. The project will provide new approaches to studying the development of AHL or provide new theories guiding the intervention of AHL on human.

A/J是典型的增龄性聋小鼠模型，新近发现其听力减退与柠檬酸合成酶(CS)基因突变有关。前期结果显示，A/J出生后第一天内耳即有高水平的Caspases表达，泛Caspases抑制剂能降低A/J的听力阈值；体外降低HEI-OC1耳蜗细胞Cs-mRNA水平能显著提高Caspase3的表达；CS是三羧酸循环的关键酶, 预测Cs基因突变导致的内质网应激、线粒体代谢障碍和最终的细胞凋亡在A/J小鼠的听力减退中起重要作用。拟在耳蜗细胞株表达Cs突变基因，观察变异蛋白（H55N)在内质网和线粒体的异常分布和引起的内质网应激；以RNAi技术建立CS低表达的耳蜗细胞模型，研究CS不足所致线粒体代谢与氧化应激标志物的变化；探讨内质网应激和线粒体损害相关凋亡途径的活化及其调控机制；在体证实关键指标并做靶向药物的干预。旨在揭示Cs突变致A/J小鼠听力减退的机制，为增龄性聋发病机制的研究提供新途径或防治提供新理论。

A/J小鼠是增龄性聋的动物模型,柠檬酸合成酶(CS)基因的错义突变(H55N)是导致A/J小鼠听力损害的主要因素。我们前期研究表明,耳蜗细胞变性在A/J小鼠的听力损害中起重要作用。本研究进一步发现:（1）Cs基因错义突变(H55N)导致CS酶活性降低约一半: 通过构建携带不同Cs基因突变序列的病毒载体,感染HEI-OC1耳蜗细胞,获得CS-Flag表达的细胞模型,发现三种形式的CS酶活性分别为100%(CS-55WT)、47%(CS-H55N)和6%(CS-d55,第55位后截断)。Cs基因错义突变导致CS酶活性降低约一半。(2)CS活性降低导致的过氧化损伤和细胞凋亡可能参与了A/J小鼠的听力损伤:利用RNA干涉技术,建立Cs基因表达下调的细胞模型(293T和HEI-OC1),发现CS表达量下降可导致细胞ATP生成减少,细胞SOD2、MitoSOX、BIP、CHOP和caspases等水平升高。进一步研究表明,CS表达量降低导致ATF6介导的内质网应激,抗氧化剂ALA可缓解CS降低对细胞的损伤作用。(3）抗氧化和抗凋亡剂(ALA和NGF)能减轻A/J小鼠的听力损害：给出生后7天的A/J小鼠肌肉注射mNGF,发现从6周龄到8周龄,治疗组小鼠ABR阈值较对照组显著性降低;6周龄时,治疗组较对照组小鼠螺旋神经元缺失显著性减少;8周龄时,治疗组小鼠毛细胞缺失亦显著性减少;从第2周龄到8周龄,治疗组小鼠内耳凋亡相关基因（Bak,Caspases等）的表达显著性下降。同时还发现α-硫辛酸可能通过减少A/J小鼠耳蜗细胞的氧化损伤,从而一定程度地减轻毛细胞和螺旋神经元的缺失,延缓A/J小鼠增龄性聋的发展进程。本研究的结果对揭示增龄性聋的发生机制有重要意义,可对其防治提供新途径或新靶点。