突变型Ubqln2促ALS运动神经元死亡的机制研究

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegerative disease characterized by motor neuron degeneration and secondary Amyotrophic lateral sclerosis (ALS) is a progressive neurodegerative disease characterized by motor neuron degeneration and secondary neurogenic muscular atrophy.It has a rapid progress and a high mortallity.With its unestablished pathogenesis,there has not been effective treatment yet.It is found that some of the ALS patients carry the mutant of Ubqln2 gene, encoding for Ubqln2 protein,which plays an important role in the formation of intracellular ubiquitinated protein inclusions in ALS patients.Previous studies have shown that the overexpressions of mutant Ubqln2 transgene in rat models can cause death of motor neurons and the inactivation of aggregated proteasome component Rpt1. We then assume that "the mutant Ubqln2 promotes Rpt1 to aggregate and then, affect the integrity and function of the proteasome, thus interfere the degradation of protein, leading to the imbalance of proteins in the brain and the development of ALS ultimately". To verify the hypothesis, we have designed this project which combines both cell experiments and animal experiments closely, to analyze the neurons and proteasome changes in mutant-type and wild-type Ubqln2 transgenic rats,explore the pathogenesis of mutant Ubqln2, looking for the relation between mutant Ubqln2 and proteasome component Rpt1 and proteasome.It is promised to find a new treatment target of ALS.

肌萎缩侧索硬化(ALS)是一种以进行性运动神经元变性和继发性神经源性肌萎缩为主要病理特征的神经退行性疾病，病情进展快，致死率高，其发病机制不明。部分ALS患者携带突变的Ubqln2基因，其编码的Ubqln2蛋白对ALS患者细胞内泛素化蛋白质包涵体的形成具有关键作用。前期研究发现过表达突变型Ubqln2转基因引起大鼠运动神经元死亡和蛋白酶体成分Rpt1出现聚集失活。由此推测“突变型Ubqln2能够促使Rpt1聚集而影响蛋白酶体的完整性和功能，引起蛋白的降解过程受阻，进而导致脑内蛋白质平衡紊乱促进ALS的发生发展”。为验证该假说，利用不同的Ubqln2动物和细胞模型，分析突变型和野生型Ubqln2转基因大鼠对神经元损害和蛋白酶体功能的影响，探索突变型Ubqln2致ALS运动神经元的死亡机制，寻找突变型Ubqln2与蛋白酶体成分Rpt1和蛋白酶体的关系；为ALS的治疗提供新的靶点。

肌萎缩侧索硬化（ALS）是一种以上、下运动神经元受损为特征性表现的神经退行性疾病，主要表现为进行性肌无力和萎缩，多种发病机制在ALS的发生发展中发挥重要作用。其中Ubqln2蛋白对ALS患者细胞内泛素化蛋白质包涵体的形成具有关键作用，为明确突变型Ubqln2是否能够促使Rpt1聚集而影响蛋白酶体的完整性和功能，引起蛋白的降解过程受阻，进而导致脑内蛋白质平衡紊乱促进ALS的发生发展。通过体内实验观察到过表达野生型和突变型Ubqln2均能够促进大鼠运动神经元的死亡，同时发现野生型和突变型Ubqln2动物脑和脊髓组织中Rpt1的表达减少，存在异常泛素化蛋白质包涵体的形成。利用蛋白质组学技术寻找Ubqln2对Rpt1的作用区域及互作位点，发现突变型Ubqln2能够促使Rpt1形成聚集和有功能的Rpt1明显减少，说明突变型的Ubqln2蛋白能够通过诱导Rpt1形成聚集体进而损害蛋白酶体的完整性。在体外实验部分，进行野生型、突变型Ubqln2和tTA转基因大鼠的皮层和运动神经元原代培养，发现突变型Ubqln2和tTA转基因大鼠的皮层和运动神经元内存在明显细胞内自噬和蛋白聚集。同时，采用蛋白质组学技术分离可溶性和不可溶性蛋白，发现神经元内存在Rpt1活性减少并形成异常聚集体。总的来说，体外实验同样证明了突变型Ubqln2能够诱导Rpt1功能减退并损害蛋白酶体的完整性。以上结果有助于深入理解ALS的发病机制并对治疗提供新的方向和思路。