解剖学报

解剖学报 ›› 2021, Vol. 52 ›› Issue (4): 520-527.doi: 10.16098/j.issn.0529-1356.2021.04.004

• 神经生物学 • 上一篇    下一篇

CTNND2基因敲除对小鼠小脑发育及运动功能的影响

王路义1 徐蔓1 汤博诣2 张潇月3 汪江杭2 王钰银2 谢乐静4 李英博1*   

  1. 1.重庆医科大学基础医学院生理学教研室; 2.第二临床学院2019级医学影像学本科; 3.第一临床学院2017级临床医学本科; 4.第二临床医学院2020级临床医学本科，重庆 400016
  • 收稿日期:2020-11-23 修回日期:2021-02-12 出版日期:2021-08-06 发布日期:2021-08-06
  • 通讯作者: 李英博 E-mail:3594833@qq.com
  • 基金资助:
    重庆医科大学创新实验项目重点项目

Effects of CTNND2 knockout on cerebellar development and motor function in mice

WANG Lu-yi1 XU Man1 TANG Bo-yi2 ZHANG Xiao-yue3 WANG Jiang-hang2 WANG Yu-yin2 XIE Le-jing4 LI Ying-bo1*   

  1. 1.Department of Physiology, School of Basic Medical Sciences;2.Medical Imaging, Grade 2019, the Second Clinical Medical College;3.Clinical Medicine, Grade 2017,the First Clinical Medical College;4. Clinical Medicine, Grade 2020, the Second Clinical Medical College, Chongqing Medical University, Chongqing 400016, China
  • Received:2020-11-23 Revised:2021-02-12 Online:2021-08-06 Published:2021-08-06
  • Contact: LI Ying-bo E-mail:3594833@qq.com

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摘要：

目的  探讨CTNND2基因敲除对小鼠小脑神经元发育和运动功能的影响及可能的机制。   方法  小鼠分为2组，每组10只，均为7周龄：野生型 (WT) C57BL/6J 小鼠为对照组，CTNND2基因敲除纯合子 (CTNND2-/-) 小鼠为实验组，PCR检测CTNND2-/- 小鼠的基因型。平衡木实验、悬挂实验和步态分析实验检测两组运动功能；免疫荧光染色、高尔基染色检测小脑普肯耶细胞的变化；Western blotting检测突触相关蛋白磷酸化突触蛋白1（p-Syn1）、突触蛋白1（Syn1）、ELKS和突触后致密蛋白 95（PSD95）以及磷脂酰肌醇-3激酶（PI3K）、磷酸化蛋白激酶B（p-Akt）、蛋白激酶B（Akt）、磷酸化哺乳动物雷帕霉素靶蛋白（p-mTOR）和哺乳动物雷帕霉素靶蛋白（mTOR）的表达水平。   结果  与WT组相比，CTNND2-/-小鼠通过平衡木的时间延长，通过平衡木的比例下降，悬挂时间缩短，悬挂得分减少，前爪步幅长度和后爪步幅长度均缩短；CTNND2-/-小鼠小脑中普肯耶细胞数及其树突的分支数均减少；p-Syn1/Syn1、p-Akt/Akt和p-mTOR/mTOR比值以及ELKS、PSD95和PI3K表达水平均低于WT组。   结论  CTNND2基因敲除可能通过下调PI3K/Akt/mTOR 信号通路，影响小脑普肯耶细胞的数量和树突结构以及突触相关蛋白的合成，导致小脑发育障碍，从而影响小鼠的运动功能。

关键词: CTNND2, 基因敲除, 小脑, 普肯耶细胞, 运动功能, 高尔基染色, 免疫荧光, 小鼠

Abstract:

Objective  To investigate the effects of CTNND2 knockout on cerebellar neuronal development and motor function in mice, as well as its possible mechanisms.    Methods  The mice were divided into two groups (n=10 in each group), all of them were 7 weeks old: wild-type (WT) C57BL/6J mice were treated as control group, and homozygous of CTNND2 knockout (CTNND2-/-) mice were treated as experimental group, the genotype of CTNND2-/-mice were detected with PCR. The motor function of two groups were detected by beam walking test, hanging wire test and gait analysis test. The changes of cerebellar Purkinje cells were detected by immunofluorescence staining and Golgi staining. Western blotting was performed to detect the expression levels of synapse-associated proteins phosphorylated synapsin 1 (p-Syn1), synapsin 1 (Syn1), ELKS and postsynaptic density protein 95(PSD95), as well as phosphoinositide 3-kinase (PI3K), phosphorylated protein kinase B (p-Akt), protein kinase B (Akt), phosphorylated mammalian target of rapamycin (p-mTOR) and mammalian target of rapamycin(mTOR).    Results  Compared with the WT mice, except the increase in time to traverse the beam, there was a decrease in the proportion of pass on the beam, or latency to fall from the hanging wire, or score of hanging wire, or fore-stride length and hind-stride length of CTNND2-/-mice. There was also a decrease in numbers of Purkinje cells and its dendritic arborization in cerebellum of CTNND2-/-mice. The ratio of p-Syn1/Syn1, p-Akt/Akt and p-mTOR/mTOR, as well as the expression levels of ELKS, PSD95 and PI3K were lower than those of WT mice.    Conclusion  CTNND2 knockout can affect the number and dendritic architecture of Purkinje cells, as well as synthesis of synapse-associated proteins in cerebellum by down-regulating PI3K/Akt/mTOR signaling pathway, resulting in cerebellar developmental disorder, thereby affecting motor function of mice.

Key words: CTNND2, Gene knockout, Cerebellum, Purkinje cell, Motor function, Golgi staining, Immunofluorescence, Mouse

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