基于ZFN技术Gcdh-/-大鼠模型的建立及其神经退行性变时Parthanatos通路的分子调控机制

Glutaric aciduria type I (GA-1) is a single gene inherited disorder caused by the accumulation of GCDH gene mutation-induced metabolites, which results in a serious harm to the infants and young children' brain development and brings heavy psychological and economic burden to the affected family as well as on all society. In previous study, it is difficult to simulate the complex pathophysiological steps of GA-I using the existing research models: primary neuron culture, tissue homogenates, subcutaneous injection in rat and Gcdh-/- mice. Domestic and foreign scholars' understanding to this disease's pathogenesis has not yet unified. Therefore, the current pressing problem is to create new cells and animal models which can simulate GA-1's disease states and then to clarify the molecular mechanism of GA-I induced neuropathological damage. Our preliminary studies showed: ①GA can damage the mitochondrial function and induce the cell apoptosis, which will lead to the damage of neuron; ②GA activates the poly [ADP-ribose] polymerase 1 (PARP-1) transcription and promotes the AIF expression, which can cause the activation of parthanatos pathway and neural degeneration. Herein, our hypothesis is: to construct GCDH gene-knockout rat GA-1 model using ZFN technology; to explore the Parthanatos pathway's molecular process that is activated by GA and other metabolic accumulation; to screen the clinical intervention target which can provide a theoretical basis for GA-1 treatment; at the same time, to provide new ideas for studying the mechanism of such single gene disorders.

戊二酸尿症I型(GA I)是GCDH基因突变致代谢产物蓄积的单基因遗传病，严重危害婴幼儿脑发育，给家庭和社会带来沉重的心理和经济负担。既往以原代神经元、组织匀浆液、皮下注射大鼠和Gcdh-/-小鼠为研究模型，难以模拟该症的复杂过程；国内外学者对本症发病机制的认识亦尚未统一。为此，创建模拟GA I疾病状态的细胞和动物模型进而探明GA I神经病理损伤的分子机制成为当前亟待解决的难题。课题组前期研究和预实验提示：①戊二酸(GA)通过损伤线粒体功能和诱导细胞凋亡致神经元损伤；②GA激活PARP-1转录，促进AIF表达，诱发parthanatos通路，启动神经退行性变。鉴于此，课题组设想：应用ZFN技术，制作Gcdh-/-大鼠，建立新型GA I 模型，解析GA等代谢蓄积物启动Parthanatos通路的分子过程，筛选临床干预靶点，为其治疗提供理论基础，同时为此类单基因遗传病的机制研究提供新思路。

戊二酸尿症I 型(GA I)是GCDH基因突变致代谢产物蓄积的单基因遗传病，严重危害婴幼儿脑发育，给家庭和社会带来沉重的心理和经济负担。课题组利用TALEN技术成功构建Gcdh-/-大鼠,进而成功模拟GA I疾病状态的细胞和动物模型。通过标准、高赖氨酸饲养Gcdh-/-大鼠神经细胞，发现培养液中戊二酸等代谢产物蓄积，神经元细胞死亡增加；以三种Gcdh-/-基因型大鼠为基础，通过标准、低、高赖氨酸饮食饲养Gcdh-/-大鼠，高赖氨酸饮食成功诱导Gcdh-/-大鼠急病危象发生，高剂量组诱导急性纹状体损伤与患儿存在一定程度的相似。在饮食诱导Gcdh-/-大鼠模型基础上，顺利对其神经损伤的分子机制进行研究，发现随GA等代谢物蓄积增加，PARP-1表达增加。同时，PARP-1抑制剂能减弱GA等代谢物对神经细胞的损伤，进而提示Parthanatos 死亡通路核心分子PARP-1参与GA I神经退行性变，PARP-1抑制剂具有减弱Gcdh-/-大鼠代谢蓄积物触发Parthanatos 死亡的作用。上述研究提示，GCDH基因缺失，GA等代谢蓄积物可能通过激活PARP-1分子，触发PARP-1下游通路，进而启动纹状体神经元Parthanatos 死亡。