中国神经再生研究(英文版)

中国神经再生研究(英文版) ›› 2021, Vol. 16 ›› Issue (12): 2549-2556.doi: 10.4103/1673-5374.313052

• 原著:神经损伤修复保护与再生 • 上一篇    下一篇

痉挛蛋白与坍塌反应调节蛋白3相互作用调控神经元的生长和分支

  

  • 出版日期:2021-12-15 发布日期:2021-05-17
  • 基金资助:

    国家自然科学基金项目（31900691，81771331，81971165）；国家基础研究计划（973计划）（2014CB542205）；广东省自然科学基金项目（2017A030313595）；广州市科学技术计划项目（201707010370）；广东省教委项目（2018KQNCX013）；中央高校基础研究基金项目（21618304）；广东省重点研究开发项目“精密医学与干细胞”重大科技专项（3242001）；中国博士后基金（2019M653292）

Spastin interacts with collapsin response mediator protein 3 to regulate neurite growth and branching

Zhi-Sheng Ji1, #, Jian-Ping Li2, #, Chao-Hua Fu1, 3, #, Jian-Xian Luo1, Hua Yang1, Guo-Wei Zhang1, Wutian Wu2, 4, 5, *, Hong-Sheng Lin1, *   

  1. 1Department of Orthopedics, The First Affiliated Hospital of Jinan University, Guangzhou, Guangdong Province, China; 2Guangdong-Hong Kong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, Guangdong Province, China; 3Department of Orthopedics, Jiangmen Hospital of Sun Yat-sen University, Jiangmen, Guangdong Province, China; 4Re-Stem Biotechnology Co., Ltd., Suzhou, Jiangsu Province, China; 5Spine Surgery, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong Province, China
  • Online:2021-12-15 Published:2021-05-17
  • Contact: Wutian Wu, MD, PhD, wtwu@hkucc.hku.hk; Hong-Sheng Lin, MD, PhD, tlinhsh@jnu.edu.cn.
  • Supported by:
    This work was supported by the National Natural Science Foundation of China, Nos. 31900691 (to GWZ), 81771331 (to HSL) and 81971165 (to WW); the National Basic Research Program of China (973 Program), No. 2014CB542205 (to WW); the Natural Science Foundation of Guangdong Province of China, No. 2017A030313595 (to HSL); the Science and Technology Program of Guangzhou, China, No. 201707010370 (to HSL); Project of Educational Commission of Guangdong Province of China, No. 2018KQNCX013 (to ZSJ); the Fundamental Research Funds for the Central Universities Project, China, No. 21618304 (to GWZ); Guangdong Provincial Key Research and Development Program “Precision Medicine and Stem Cell” Major Science and Technology Project, China, No. 3242001 (to WW); and China Postdoctoral Science Foundation, No. 2019M653292 (to ZSJ).

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摘要：

细胞骨架微管的重新排列和运动是脊髓损伤后修复的关键环节，痉挛蛋白在调节微管切割的方面起重要作用。虽然痉挛蛋白和坍塌反应调节蛋白都可调节轴突的生长和分支，但坍塌反应调节蛋白3和痉挛蛋白是否发生相互作用及坍塌反应调节蛋白3参与脊髓损伤修复的机制尚待确定。实验采用了蛋白质组学方法来鉴定与脊髓损伤修复过程相关的关键蛋白，紧接着通过液相色谱-质谱方法鉴定出坍塌反应调节蛋白是能与谷胱甘肽S-转移酶-痉挛蛋白相互作用的潜在蛋白，且通过使用免疫共沉淀和免疫荧光等确定了痉挛蛋白与坍塌反应调节蛋白3之间存在相互作用，最后用坍塌反应调节蛋白3和痉挛蛋白共同转染海马神经元。结果显示：坍塌反应调节蛋白3在脊髓损伤过程中发挥了作用，液相色谱-质谱下拉法确定了3种能够与坍塌蛋白起相互作用的坍塌反应调节蛋白；继而发现坍塌反应调节蛋白3和痉挛蛋白可在脊髓中共表达，且这2种蛋白能够在体内外发生相互作用；最后发现坍塌反应调节蛋白3过表达能够增强痉挛蛋白促进神经突生长和分支的能力；因此证实了痉挛蛋白和坍塌反应调节蛋白3通过调节神经突生长和分支生成在脊髓损伤和修复方面发挥重要作用，且这些蛋白可能是治疗脊髓损伤的新靶点。实验于2018年经暨南大学动物伦理委员会批准（批准号IACUS-20181008-03）。

https://orcid.org/0000-0002-6945-8875 (Hong-Sheng Lin)

https://orcid.org/0000-0001-2345-6789 (Wutian Wu)

关键词: 痉挛蛋白, 坍塌反应调节蛋白3, 蛋白相互作用, 脊髓损伤, 液相色谱-质谱, 微管, 神经突生长, 蛋白质组学

Abstract: Cytoskeletal microtubule rearrangement and movement are crucial in the repair of spinal cord injury. Spastin plays an important role in the regulation of microtubule severing. Both spastin and collapsin response mediator proteins can regulate neurite growth and branching; however, whether spastin interacts with collapsin response mediator protein 3 (CRMP3) during this process remains unclear, as is the mechanism by which CRMP3 participates in the repair of spinal cord injury. In this study, we used a proteomics approach to identify key proteins associated with spinal cord injury repair. We then employed liquid chromatography-mass spectrometry to identify proteins that were able to interact with glutathione S-transferase-spastin. Then, co-immunoprecipitation and staining approaches were used to evaluate potential interactions between spastin and CRMP3. Finally, we co-transfected primary hippocampal neurons with CRMP3 and spastin to evaluate their role in neurite outgrowth. Mass spectrometry identified the role of CRMP3 in the spinal cord injury repair process. Liquid chromatography-mass spectrometry pulldown assays identified three CRMP3 peptides that were able to interact with spastin. CRMP3 and spastin were co-expressed in the spinal cord and were able to interact with one another in vitro and in vivo. Lastly, CRMP3 overexpression was able to enhance the ability of spastin to promote neurite growth and branching. Therefore, our results confirm that spastin and CRMP3 play roles in spinal cord injury repair by regulating neurite growth and branching. These proteins may therefore be novel targets for spinal cord injury repair. The Institutional Animal Care and Use Committee of Jinan University, China approved this study (approval No. IACUS-20181008-03) on October 8, 2018. 

Key words: collapsin response mediator protein 3, liquid chromatography-mass spectrometry, microtubule, neurite growth, protein interactions, proteomics, spastin, spinal cord injury

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