核蛋白TAR DNA/RNA结合蛋白43与小鼠肌萎缩侧索硬化症的关系

Relationship between nucleoprotein TAR DNA/RNA binding protein 43 and mouse amyotrophic lateral sclerosis

目的利用HB9启动子构建小鼠脊髓运动神经元特异表达人类TAR DNA/RNA结合蛋白43(hTDP-43)突变转基因小鼠,建立肌萎缩侧索硬化症(ALS)疾病模型,探究hTDP-43突变导致ALS发生的机制。方法体外构建HB9启动子连接突变hTDP-43载体,通过原核注射制备并筛选阳性转基因小鼠品系(Q331K位点和M337V位点突变各8~10只)。通过步态分析、转棒疲劳仪实验和悬挂实验检测小鼠运动能力;通过免疫组织化学法、免疫荧光染色和Western blotting分别检测hTDP-43、磷酸化hTDP-43(p-hTDP-43),Caspase-3、剪切Caspase-3(cleaved Caspase-3)、泛素蛋白、β-微管蛋白Ⅲ(Tuj1)、Ki67和细胞周期依赖性激酶5(CDK5)蛋白的表达情况。结果脊髓运动神经元表达突变hTDP-43蛋白的转基因小鼠双后肢向躯干侧回缩,运动机能随月龄增加呈现进程性减退。转基因小鼠脊髓运动神经元可见hTDP-43,p-hTDP43,Caspase-3,cleaved Caspase-3阳性染色和泛素蛋白阳性包涵体,体外分离培养脊髓胸腰段运动神经元发现hTDP-43和泛素蛋白共定位于胆碱乙酰基转位酶(ChAT)阳性的运动神经元,并伴随CDK5异位表达。结论小鼠脊髓运动神经元表达突变的hTDP-43蛋白,可促使分化的成熟神经元重新进入细胞周期,导致ALS发生。

Objective Transgenic mice expressing human TAR DNA/RNA binding protein 43(hTDP-43)mutant protein in spinal cord motor neurons were constructed using HB9 promoter to establish a disease model of amyotrophic lateral sclerosis(ALS)and explore the mechanism of ALS induced by hTDP-43 mutation.Methods HB9 promoter junction mutant hTDP-43 vector was constructed in vitro,and the positive transgenic mouse strains were prepared by prokaryotic injection and screened(There were 8~10 mutations at Q331K and M337V).Gait analysis,rotary rod fatigue test,and suspension test were used to detect locomotion ability of mice.Immunohistochemistry,immunofluorescence staining and Western blotting were used to detect hTDP-43,phosphorylated HTDP-43(p-hTDP-43),Caspase-3,cleaved Caspase-3,respectively.Expression of ubiquitin,β-tubulinⅢ(Tuj1),Ki67 and cyclin-dependent kinase 5(CDK5)proteins were also detected.Results In transgenic mice expressing mutant hTDP-43 protein in spinal motor neurons,both hind limbs were atrophied to the trunk side,and motor function showed progressive decline with increasing age.hTDP-43,p-hTDP-43,Caspase-3,and cleaved Caspase-3 were observed in spinal motor neurons Caspase-3 positive staining and ubiquitin protein positive inclusion body,and in vitro isolation and culture of spinal motor neurons,it was found that hTDP-43 and ubiquitin protein co-located in choline acetyl translocation enzyme(ChAT)positive motor neurons,accompanied by ectopic expression of CDK5.Conclusion The mutant HDP 43 protein expressed in mouse spinal cord motor neurons can promote the re-entry of differentiated mature neurons into the cell cycle,leading to the occurrence of ALS..