中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2020, Vol. 15 ›› Issue (12): 2335-2343.doi: 10.4103/1673-5374.285002

• 原著:脑损伤修复保护与再生 • 上一篇    下一篇

胰岛素样生长因子1部分修复了SHANK2敲低人类神经元的早期发育缺陷

  

  • 出版日期:2020-12-15 发布日期:2020-08-05
  • 基金资助:

    广东省资助的“治疗脑部疾病的关键技术”（2018B030332001）；中国广州卫生与医疗合作创新重大项目（201803040016-2，201604046028）；中国广东省科学技术计划和关键技术创新项目（2014B050504006），中国大学学科引进人才计划（B14036）；中国广东省科学技术计划项目（2017B090904033）。

Insulin-like growth factor 1 partially rescues early developmental defects caused by SHANK2 knockdown in human neurons

Shu-Ting Chen 1 , Wan-Jing Lai 1, 2 , Wei-Jia Zhang 1 , Qing-Pei Chen 1 , Li-Bing Zhou 1 , Kwok-Fai So 1 , Ling-Ling Shi 1, 3, 4   

  1. 1 Guangdong-Hong Kong-Macau Institute of CNS Regeneration, Ministry of Education CNS Regeneration Collaborative Joint Laboratory, Jinan University, Guangzhou, Guangdong Province, China
    2 Clinical Medicine, Jinan University, Guangzhou, Guangdong Province, China
    3 Department of Psychiatry, the First Affiliated Hospital of Jinan University, Guangzhou, Guangdong Province, China
    4 Co-innovation Center of Neuroregeneration, Nantong University, Nantong, Jiangsu Province, China
  • Online:2020-12-15 Published:2020-08-05
  • Contact: Ling-Ling Shi, MD, PhD,tlingshi@jnu.edu.cn.
  • Supported by:
    This study was supported by the National Natural Science Foundation of China, No. 81771222 (to LLS); the Natural Science Foundation of Guangdong Province of China, No. 2019A1515011316 (to LLS); the National Key Research and Development Program of China, Stem Cell and Translational Research, No. 2017YFA0105102 (to LLS); the Guangzhou Science and Technology Innovation Development Special Fund Project of China, No. 201804010212 (to LLS); the Program of Introducing Talents of Discipline to Universities of China, No. B14036 (to KFS).

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摘要：

SHANK2是兴奋性突触后致密区的支架蛋白，且SHANK2基因变异与突触功能障碍，自闭症谱系障碍的病理生理致病机制密切相关。近期已有研究提示神经元早期发育异常可能参与自闭症谱系障碍的病理生理过程，也有研究提示胰岛素样生长因子1(IGF1)对神经元突起发育有正性作用。为探索SHANK2表达下调对神经元早期发育的影响，实验建立了一个基于多能干细胞诱导分化为神经元的稀疏培养系统。对人源诱导多能干细胞进行神经分化，分化至神经前体细胞阶段(培养3-14 d)，将其消化为单个神经元，并给予携带shRNA信息和红色荧光信号(RFP)的慢病毒进行感染，设为shSHANK2组，对照组用shControl慢病毒进行感染，设为shControl组。(1)采用感染神经元并全细胞表达的慢病毒RFP信号进行神经元胞体、突起等二维结构重建、成像检测分别评估两组谷氨酸转运体蛋白1(VGLUT1)阳性的谷氨酸神经元及络氨酸羟化酶(TH)阳性的多巴胺神经元的突起长度和复杂度变化发现，与shControl 组VGLUT1+神经元相比，shSHANK2组VGLUT1+神经元的突起长度和复杂度降低，且移动速率增快。(2)给予IGF1干预干预后的IGF1干预组比未干预组的突起长度增加和复杂度增加，但运动速率无显著改变。提示IGF1仅能逆转shSHANK2神经元的形态缺陷而非运动异常；(3)结果数据证实，SHANK2表达异常可影响神经元早期发育，研究也提示IGF1作为一个潜在的干预因子，可部分逆转继发于SHANK2表达异常的神经元发育障碍。

orcid: 0000-0003-4225-209X (Ling-Ling Shi)

Abstract: SHANK2 is a scaffold protein that serves as a protein anchor at the postsynaptic density in neurons. Genetic variants of SHANK2 are strongly associated with synaptic dysfunction and the pathophysiology of autism spectrum disorder. Recent studies indicate that early neuronal developmental defects play a role in the pathogenesis of autism spectrum disorder, and that insulin-like growth factor 1 has a positive effect on neurite development. To investigate the effects of SHANK2 knockdown on early neuronal development, we generated a sparse culture system using human induced pluripotent stem cells, which then differentiated into neural progenitor cells after 3–14 days in culture, and which were dissociated into single neurons. Neurons in the experimental group were infected with shSHANK2 lentivirus car- rying a red fluorescent protein reporter (shSHANK2 group). Control neurons were infected with scrambled shControl lentivirus carrying a red fluorescent protein reporter (shControl group). Neuronal somata and neurites were reconstructed based on the lentiviral red fluo- rescent protein signal. Developmental dendritic and motility changes in VGLUT1 + glutamatergic neurons and TH + dopaminergic neurons were then evaluated in both groups. Compared with shControl VGLUT1 + neurons, the dendritic length and arborizations of shSHANK2 VGLUT1 + neurons were shorter and fewer, while cell soma speed was higher. Furthermore, dendritic length and arborization were sig- nificantly increased after insulin-like growth factor 1 treatment of shSHANK2 neurons, while cell soma speed remained unaffected. These results suggest that insulin-like growth factor 1 can rescue morphological defects, but not the change in neuronal motility. Collectively, our findings demonstrate that SHANK2 deficiency perturbs early neuronal development, and that IGF1 can partially rescue the neuronal defects caused by SHANK2 knockdown. All experimental procedures and protocols were approved by the Laboratory Animal Ethics Com- mittee of Jinan University, China (approval No. 20170228010) on February 28, 2017.

Key words: cells, factor, growth, in vitro, model, neural differentiation, neurogenesis, plasticity