CRMP5通过Spastin释放微管动力促进神经突起分支形成

发布时间：2018-05-07 20:08

本文选题：Spastin + CRMP5　；参考：《暨南大学》2017年博士论文

【摘要】：目的:神经网络的建立需神经突起的生长发育提供物质基础,而神经突起的生长发育主要通过改变细胞骨架的运动进行调控,细胞骨架的运动主要依靠微管与微丝间的不断聚合和解聚,而微管与微丝间的互作机制仍未完全阐明。微管切割蛋白Spastin与微管结合蛋白CRMP5均参与神经突起生长过程,前期我们已证实Spastin与C RMP5能相互结合并明确相互结合位点,本研究的主要目的在于进一步明确Spastin协同CRMP5如何介导细胞骨架的运动,调控神经突起的生长发育。材料与方法:首先通过免疫荧光染色,在共聚焦显微镜下观察Spastin与CRMP5在Hela细胞和神经元中共定位的情况;在Hela细胞中过表达Spastin~(M87V)及各截短片并结合共聚焦显微镜观察微管的切割情况;培养原代神经元,并利用磷酸钙转染方法导入Spastin~(M87V)及各截短片,观察神经元突起的变化情况;进一步我们设计针对Spastin相对特异的干扰小片段si-Spastin导入神经元中并观察对其突起的影响情况;将Spastin~(M87V)及各截短片导入神经元中并记录m EPSC的变化情况;同样,在原代神经元中转染CRMP5观察神经元突起的变化情况;我们进一步在Hela细胞中共转染Spastin~(M87V)和CRMP5,观察微管切割的变化;同时,在神经元中共转染Spastin~(M87V)和CRMP5,观察神经元突起的变化情况;通过设计不同的分组将质粒导入神经元中,分别观察同时沉默Spastin与CRMP5以及沉默Spastin而过表达CRMP5或沉默CRMP5而过表达Spastin不同处理对神经元突起的影响情况;在神经元中进一步共转染Spastin~(M87V)和CRMP5,观察m EPSC的变化情况。结果:1)免疫细胞化学结果示:Spastin与CRMP5共存于Hela细胞的胞质,神经元的胞质和突起中。2)培养Hela细胞并过表达Spastin~(M87V)、Spastin N190、Spastin△AAA、Spastin△N1、Spastin△N2基因:Spastin~(M87V)和Spastin△N1基因能将Hela细胞微管切割成小片段。3)培养原代海马神经元并过表达Spastin~(M87V)、Spastin N190、Spastin△AAA、Spastin△N1、Spastin△N2:Spastin~(M87V)、Spastin△N1、Spastin△N2可以明显促进海马神经元轴突、树突侧枝的形成和突起生长。4)将GFP、Spastin~(M87V)、Spastin N190、Spastin△AAA、Spastin△N1、Spastin△N2导入神经元中,用全细胞膜片钳检测结果示:Spastin~(M87V)、Spastin△N1、Spastin△N2组神经元的m EPSC的频率和幅度均较GFP+m C herry组增大。5)设计Spastin si-RNA干扰片段,与Spastin~(M87V)共导入293T细胞中,western-blot检测其干扰效果明显,导入神经元,观察到干扰Spastin对神经突起的生长有抑制作用。6)在原代海马神经元过表达CRMP5、CRMP5N471、CRMP5△471结果示:CRMP5、CRMP5△471可以明显促进海马神经元轴突、树突侧枝的形成和突起生长。7)在Hela细胞共转染GFP和m Cherry、Spastin~(M87V)和CRMP5、Spastin~(M87V)和m C herry、GFP和CRMP5结果示:导入CRMP5不影响Spastin~(M87V)对Hela细胞微管的切割效率。8)在原代海马神经元共过表达GFP和Mecherry、Spastin~(M87V)和CRMP5、Spastin~(M87V)和m Cherry、GFP和CRMP5结果示:Spastin~(M87V)和CRMP5组促进海马神经元轴突、树突侧枝的形成和突起生长更加明显。9)在神经元中分别导入NC、si-Spastin、si-CRMP5、si-Spastin+si-CRMP5、si-Spastin+C RMP5、Spastin+si-CRMP5结果示:干扰Spastin或C RMP5均抑制神经元突起的生长,同时干扰两者则抑制神经元突起生长更明显,若干扰Spastin或CRMP5,同时过表达Spastin或CRMP5则能部分抵消抑制突起生长作用。10)将GFP+m C herry、Spastin~(M87V)+CRMP5、Spastin~(M87V)+m C herry和CRMP5+GFP导入神经元,用全细胞膜片钳检测结果示:Spastin~(M87V)+CRMP5、Spastin~(M87V)+m C herry、CRMP5+GFP组的m EPSC的频率和幅度均较GFP+m C herry组增大。结论:1)Spastin与C RMP5在体内存在相互共定位。2)Spastin~(M87V)对Hela细胞的微管的切割作用需有完整的微管结合区(MTBD区)和AAA ATP催化区(AAA区)。3)过表达CRMP5不影响Spastin~(M87V)对Hela细胞微管的切割效率。4)Spastin~(M87V)对神经元突起生长及分支形成的促进作用与MTBD及AAA功能域密切相关。5)CRMP5对神经元突起生长及分支形成促进作用的主要功能域在472-564氨基酸间。6)Spastin~(M87V)与CRMP5能协同调控神经元突起生长及分支的形成。
[Abstract]:Objective: the establishment of neural networks requires a material basis for the growth and development of neurites, and the growth and development of the neurites are regulated mainly by the movement of the cytoskeleton. The movement of the cytoskeleton depends on the continuous polymerization and disaggregation between microtubules and microfilaments, and the interaction mechanism between microtubules and microfilaments is still not fully elucidated. Microtubule cuts Spastin and microtubule binding protein CRMP5 are both involved in the process of neurite growth. In the early stage, we have confirmed that Spastin and C RMP5 can be combined with each other and define the binding sites. The main purpose of this study is to further clarify how Spastin and CRMP5 can mediate the transport of cytoskeleton and regulate the growth and development of neurites. Methods: Spastin and CRMP5 were observed by immunofluorescence staining under confocal microscopy in Hela cells and neurons. Spastin~ (M87V) and short films were overexpressed in Hela cells and the microtubule cutting was observed with confocal microscopy, the primary neurons were cultured and Spast was transfected into Spast by calcium phosphate transfection method. In~ (M87V) and the short clips to observe the changes in the neurite protuberance; further we design a relatively specific Spastin interfering small fragment of si-Spastin into the neuron and observe its influence on its protuberance; Spastin~ (M87V) and each short film are introduced into neurons and the changes of M EPSC are recorded; the same, in the primary neurons CRMP5 was used to observe the changes in the neurites of the neurons. We further transfected Spastin~ (M87V) and CRMP5 in Hela cells to observe the changes in microtubule cutting. At the same time, we transfected Spastin~ (M87V) and CRMP5 in the neurons to observe the changes in the neurite protuberance, and the plasmid was introduced into neurons by designing different groups. The effects of simultaneous silence of Spastin and CRMP5 and silence of Spastin on the expression of CRMP5 or silent CRMP5 and the effects of Spastin on the neuronal protuberance were observed; Spastin~ (M87V) and CRMP5 were further co transfected in neurons, and the changes in M EPSC were observed. Results: 1) the immunocytochemical results showed that Spastin was coexisting with the M. Spastin~ (M87V), Spastin N190, Spastin Delta AAA, Spastin Delta N1, Spastin Delta N2 genes are expressed in the cytoplasm of the cell, the cytoplasm of the neuron and the.2 in the protuberance. N Delta AAA, Spastin Delta N1, Spastin Delta N2:Spastin~ (M87V), Spastin Delta N1, Spastin Delta N2 can obviously promote hippocampus neuron axon, dendritic lateral branch formation and initiation growth.4). The frequency and amplitude of M EPSC of pastin Delta N1, Spastin Delta N2 group neurons are more than GFP+m C Herry group.5) designed Spastin si-RNA interference fragment. The overexpression of CRMP5, CRMP5N471, CRMP5 delta 471 shows that CRMP5, CRMP5 delta 471 can obviously promote the axon of hippocampal neurons, the formation of dendrites and the growth of.7) in the Hela cells co transfected with GFP and m Cherry. Hela cell microtubule cutting efficiency.8) in the primary hippocampal neurons, the expression of GFP and Mecherry, Spastin~ (M87V) and CRMP5, Spastin~ (M87V) and m Cherry, GFP, and m Cherry, the formation of the hippocampal neurons, the formation of the dendrites and the growth of the protuberance are more obvious. Si-CRMP5, si-Spastin+si-CRMP5, si-Spastin+C RMP5, Spastin+si-CRMP5 results show that interference of Spastin or C RMP5 all inhibit the growth of neuronal protuberance, while interference both inhibits the growth of neuron protuberances more clearly, if Spastin or CRMP5 is interfered with, while overexpression Spastin or CRMP5 can partially counteract the inhibition of protuberance growth Herry, Spastin~ (M87V) +CRMP5, Spastin~ (M87V) +m C Herry and CRMP5+GFP import neurons. 87V) cutting effect on microtubules of Hela cells requires a complete microtubule binding zone (MTBD region) and AAA ATP catalytic region (AAA region).3) over expression CRMP5 does not affect the efficiency of Spastin~ (M87V) cutting efficiency of Hela cell microtubules. The main functional domain of neurite protuberance growth and branching formation is.6 between 472-564 amino acids.) Spastin~ (M87V) and CRMP5 can regulate the growth of neurites and the formation of branches in synergy.

【学位授予单位】：暨南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R338

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