解剖学报

解剖学报 ›› 2022, Vol. 53 ›› Issue (4): 440-446.doi: 10.16098/j.issn.0529-1356.2022.04.006

• 神经生物学 • 上一篇    下一篇

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

唐伟博1 刘丽2 申景岭2,3*   

  1. 1. 吉林医药学院肿瘤靶向治疗与转化医学实验室，吉林 吉林 132000; 2. 温州大学生命与环境科学学院生命科学研究院，浙江 温州 325035; 3. 哈尔滨医科大学组织学与胚胎学教研室，哈尔滨 150081)
  • 收稿日期:2021-11-04 修回日期:2022-01-15 出版日期:2022-08-06 发布日期:2022-09-11
  • 通讯作者: 申景岭 E-mail:20190060@wzu.edu.cn
  • 基金资助:
    建立人TDP-43突变疾病模型探讨TDP-43在ALS中的功能

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

TANG  Wei-bo1  LIU  Li2  SHEN  Jing-ling2,3*   

  1. 1.Laboratory of Targeted Therapy and Translational Medicine, Jilin Medicine University, Jilin Jilin 132000,China; 2.College of Life and Environmental Sciences, Wenzhou University, Zhejiang Wenzhou 325035, China; 3.Department of Histology and Embryology, Harbin Medical University, Harbin 150081, China
  • Received:2021-11-04 Revised:2022-01-15 Online:2022-08-06 Published:2022-09-11
  • Contact: SHEN Jing-ling E-mail:20190060@wzu.edu.cn

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摘要：

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。

关键词: TAR DNA/RNA 结合蛋白43, 肌萎缩侧索硬化症, 细胞周期依赖性激酶5, 神经退行性疾病, 脊髓运动神经元, 免疫印迹法, 小鼠

Abstract:

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.

Key words: TAR DNA/RNA binding protein 43, Amyotrophic lateral sclerosis, Cyclin-dependent kinase 5, Neurodegeneration, Spinal cord motor neuron, Western blotting, Mouse

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