β-catenin通过抑制Npc1泛素化降解调控C1型尼曼匹克病胆固醇代谢的作用和机制研究

Niemann-Pick type C1 disease (NPC1) is a progressive lysosomal lipid storage disease which seriously affecting the patient’s life. Our previous data showed that Npc1 loss can lead to down-regulation of β-catenin expression. Further, long-term feeding of Npc1 mutant mouse with LiCl can effectively reduce the accumulation of cholesterol and increase expression of Npc1 in mutants, but the regulatory mechanism is not clear. According to above, we propose that β-catenin regulates cholesterol metabolism in NPC1 disease through preventing ubiquitin-mediated degradation of Npc1 protein. In this study, we aim to investigate the function of β-catenin regulating cholesterol metabolism in vitro and in vivo through activaing and inhibiting β-catenin. Meanwhile, we will demonstrate the function and regulation of β-catenin on cholesterol metabolism through investigating accumulation of free cholesterol and related gene expression in 293T, MEF cells and the liver, cerebral cortex, spleen of Npc1 mutant mice. Finally, we will reveal functional mechanism of β-catenin inhibiting ubiquitin-mediated degradation of Npc1 in NPC1 disease.This project will provide experimental data to reveal functional mechanism of β-catenin regulates cholesterol metabolism in NPC1 disorders and its potential clinical application.

C1型尼曼匹克病（NPC1）是Npc1基因突变所导致的进行性溶酶体胆固醇贮积病，严重影响患者的生活质量。前期数据表明Npc1缺失导致β-catenin下调，进而用β-catenin激活剂LiCl喂食Npc1突变小鼠能稳定Npc1的表达并有效减少胆固醇在胞内的贮积，但具体调控机制尚不明确。据此我们推测β-catenin通过抑制Npc1泛素化降解（稳定Npc1的表达）调控胆固醇代谢。本项目拟在细胞和动物水平分别激活和抑制β-catenin，检测其对293T，MEF细胞和突变小鼠肝脏、大脑皮层、脾脏中胆固醇异常代谢的调控及胆固醇转运过程中相关基因的表达，阐明β-catenin对胆固醇代谢的调控作用；在此基础上通过半衰期和Npc1泛素化水平的测定揭示β-catenin调控Npc1泛素化降解的机制。本研究将阐释β-catenin调控NPC1病胆固醇代谢的机制，并为NPC1治疗提供新的思路。

C1型尼曼匹克病（NPC1）是Npc1基因突变所导致的进行性溶酶体胆固醇贮积病，严重影响患者的生活质量。本项目通过免疫组化/荧光、WB等技术，明确了转录因子β-catenin在Npc1突变小鼠中表达下调；利用不同的β-catenin激活剂（LiCl和CHIR99021）处理Npc1突变小鼠，证实β-catenin激活剂提高小鼠体重水平并延长寿命；进一步发现激活β-catenin可以减缓Npc1蛋白的降解，并降低胆固醇的贮积。最后初步探讨了Npc1基因缺失对内体分选系统的影响。本项目在初步完成了既定目标的前提下，研究了二甲双胍联合β环糊精（HPβCD）对Npc1小鼠的治疗作用，发现联合治疗相比较于单独HPβCD治疗并没有明显的延长寿命，提高体重；但二甲双胍治疗能够降低NPC1病灶部位（脑、肝、脾）的炎症侵润，为二甲双胍在NPC1疾病治疗提供临床前数据。