CNTN4调节APP引起视网膜神经节细胞损伤的作用机制研究

Glaucoma is a major cause of blindness characterized as progressive RGCs damage and visual field defects, which is the most important etiology of irreversible blindness worldwide. A growing number of studies have suggested that genetic basis is an important cause in POAG. It is proved that CNTN4 is a new glaucoma candidate gene in the cohort study. And our results revealed that CNTN4 was colocalized with APP in mice primary RGCs. Besides, in the retina of our chronic ocular hypertension mice model induced by photocoagulation, the protein level of CNTN4 as well as full-length APP was significant increased compared to control mice. Altogether, it is indicated that CNTN4 can lead to glaucomatous RGCs damage via interacting with APP. Yet the possible mechanism needs further study. In our project CNTN4 WT and KO mice will be used to create chronic ocular hypertension mice model and primary mice RGCs model. Biochemical, histochemical, neurobiological, and cell biological techniques will be utilized to detect the apoptosis and synapse morphology of RGCs. Moreover, APP processing, the expression and activity of secretases, axonal transportation of APP and Aβ will be analyzed. Our project is aimed to illustrate the possible mechanism that CNTN4 cause glaucomatous RGCs damage through regulating the transportation and processing of APP. It is hoped that our study will improve prevention and treatment of glaucoma.

青光眼是一类以进行性的视网膜神经节细胞（RGCs）损伤及视野缺损为特征的不可逆致盲性眼病。众多研究表明原发性开角型青光眼（POAG）发病与遗传因素有关，病例-对照研究则显示CNTN4是POAG新的候选基因。本项目前期研究证实小鼠原代RGCs中CNTN4与APP共定位，高眼压青光眼小鼠视网膜CNTN4的表达和APP的蛋白量较正常组均增加。为进一步探究CNTN4与APP相互作用造成青光眼RGCs损伤的机制，本研究拟通过激光光凝诱导CNTN4 WT和KO小鼠建立高眼压青光眼模型，并分离小鼠原代RGCs，采用生物化学、组织化学、神经生物学和细胞生物学等技术，观察RGCs的凋亡及其轴突树突等的形态，分析APP的剪切方式、剪切酶表达和活性、APP和Aβ的轴浆转运，以期揭示CNTN4调节APP的剪切和转运在青光眼RGCs损伤中的作用机制，为青光眼的防治打下坚实的基础。

项目首先利用视神经损伤的CNTN4敲除小鼠、原代RGCs和HEK293作为动物模型及细胞模型，通过对敲除小鼠RGCs计数、检测Caspase 3、视神经CTB标记，发现小鼠视网膜CNTN4的表达促进视神经损伤导致的RGCs死亡和轴突退变。利用原位杂交和WB检测视网膜发育中CNTN4和APP的表达情况，显示CNTN4和APP在视网膜发育后期及成年视网膜中发挥作用。为研究CNTN4对APP剪切的作用，对CNTN4敲除小鼠视网膜行WB检测，将CNTN4突变体转染稳定表达APP的HEK293细胞系观察APP及Aβ的蛋白量，并在RGCs中观察APP的定位及含量，发现CNTN4可促进Aβ生成，并在神经突中帮助APP保持特定细胞定位，CNTN4的SP结构域是促进APP剪切的关键结构域。对视网膜中APP剪切关键酶研究发现CNTN4有助于神经突BACE1的点状分布及突触后定位，CNTN4可维持视网膜β-分泌酶活性。为进一步阐明CNTN4介导RGCs死亡及轴突损伤的机制，对CNTN4野生型和敲除小鼠视网膜行表达谱分析，研究发现差异表达基因主要集中在代谢、线粒体组分、转录因子和蛋白结合等过程；CNTN4致神经退行性疾病相关通路发生改变，细胞能量代谢相关通路及mRNA转录翻译通路也受到影响。综上所述，CNTN4是视网膜神经损伤后RGCs死亡及APP异常剪切的因素之一，CNTN4有助于维持神经突上β-分泌酶的定位及视网膜中β-分泌酶活性；CNTN4通过参与代谢、细胞器组分（主要是线粒体）、转录因子和蛋白结合等生理过程，在神经退行性疾病、细胞能量代谢及mRNA转录翻译通路发挥促RGCs损伤的作用。因此，通过敲除/降低CNTN4表达、抑制SP结构域的功能或调节CNTN4调节的通路，有利于缓解RGCs的死亡；CNTN4蛋白、其SP结构域及下游相关通路，可作为青光眼等眼部神经退行性疾病的潜在治疗靶点。