南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (1): 78-85.doi: 10.12122/j.issn.1673-4254.2022.01.09

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RFWD2调控小鼠大脑皮层神经元树突发育及树突棘形成的功能效应

孙婷婷，王圆圆，方祝玲，徐佳佳，马世玟，常久翔，刘高峰，郭 俣，刘长青   

  1. 蚌埠医学院生命科学学院，检验医学院，临床医学院，安徽 蚌埠 233000
  • 出版日期:2022-01-20 发布日期:2022-03-02

Effects of ring finger and tryptophan-aspartic acid 2 on dendritic spines and synapse formation in cerebral cortex neurons of mice

SUN Tingting, WANG Yuanyuan, FANG Zhuling, XU Jiajia, MA Shiwen, CHANG Jiuxiang, LIU Gaofeng, GUO Yu, LIU Changqing   

  1. School of Life Sciences, School of Laboratory Medicine, School of Clinical Medicine, Bengbu Medical College, Bengbu 233000, China
  • Online:2022-01-20 Published:2022-03-02

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摘要： 目的 探讨环指和色氨酸-天冬氨酸重复序列结构域2（RFWD2）差异表达对小鼠大脑皮层神经元树突发育及树突棘形成的功能效应。方法 利用免疫荧光鉴定RFWD2在小鼠全脑的定位及表达谱，通过RFWD2与神经元突触蛋白的共定位，明确其在神经元的细胞定位。通过电转染RFWD2-Myc过表达与shRNA载体，分析其对体外培养小鼠大脑皮层神经元树突发育、树突棘形成与突触功能的影响及作用机制。结果 RFWD2在小鼠大脑皮层与海马区均高表达，与突触形成具有明显的正相关性。RFWD2在神经元的突触前膜及后膜均有表达，与神经元树突的长度、分支数量与复杂性呈正相关。与对照组相比，RFWD2 过表达显著降低了神经元突触蛋白数量［突触小泡蛋白（SYN）4.5±1.2 vs 17.2±2.4/10 μm，P<0.01；突触后致密蛋白（PSD95）：2.1±0.3 vs 8.5±1.3/10 μm，P<0.01］；而RFWD2抑制表达显著增加了突触蛋白数量（SYN：19.6±2.6/10 μm，P<0.05；PSD95：11.5±1.1/10 μm，P<0.05）。与对照组和RFWD2过表达相比，RFWD2抑制表达降低了树突棘的数量（4.3±0.2 vs 6.2±0.5/10 μm，P< 0.05；4.3±0.2 vs 6.9±1.1/10 μm，P<0.01）。结论 RFWD2可通过Pea3家族成员与c-Jun的泛素化影响突触蛋白的表达，调控小鼠大脑皮层神经元树突发育、树突棘形成与突触功能效应，为后期作为靶点开展神经系统疾病的治疗奠定基础。

关键词: RFWD2；神经元；树突棘；突触

Abstract: Objective To clarify the functional effects of differential expression of ring finger and tryptophan-aspartic acid 2 (RFWD2) on dendritic development and formation of dendritic spines in cerebral cortex neurons of mice. Methods Immunofluorescent staining was used to identify the location and global expression profile of RFWD2 in mouse brain and determine the co-localization of RFWD2 with the synaptic proteins in the cortical neurons. We also examined the effects of RFWD2 over-expression (RFWD2-Myc) and RFWD2 knockdown (RFWD2-shRNA) on dendritic development, dendritic spine formation and synaptic function in cultured cortical neurons. Results RFWD2 is highly expressed in the cerebral cortex and hippocampus of mice, and its expression level was positively correlated with the development of cerebral cortex neurons and dendrites. RFWD2 expression was detected on the presynaptic membrane and postsynaptic membrane of the neurons, and its expression levels were positively correlated with the length, number of branches and complexity of the dendrites. In cultured cortical neurons, RFWD2 overexpression significantly lowered the expressions of the synaptic proteins synaptophysin (P<0.01) and postsynapic density protein 95 (P<0.01), while RFWD2 knockdown significantly increased their expressions (both P<0.05). Compared with the control and RFWD2-overexpressing cells, the neurons with RFWD2 knockdown showed significantly reduced number of dendritic spines (both P<0.05). Conclusion RFWD2 can regulate the expression of the synaptic proteins, the development of the dendrites,the formation of the dendritic spines and synaptic function in mouse cerebral cortex neurons through ubiquitination of Pea3 family members and c-Jun, which may serve as potential treatment targets for neurological diseases.

Key words: ring finger and tryptophan-aspartic acid 2; cortical neurons; dendritic spines; synapses