Molecular mechanism of autosomal dominant hererditary spastic paraplegia type 4(SPG4)

常染色体显性遗传性痉挛性截瘫4型（SPG4）的分子机制

基本信息

批准号：
15590903
负责人：
TAKIYAMA Yoshihisa
金额：
$ 2.24万
依托单位：
Jichi Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590903/
关键词：
Hereditary spastic paraplegia Spastin AAA family protein siRNA Hereditary Spastic Paraplegia AAA family protein hereditary spastic paraplegia spastin

项目摘要

Most cases of autosomal dominant form of hereditary spastic paraplegia are due to mutations in the SPG4 gene, which encodes spastin, an ATPase belonging to the AAA family. Although spastin are suggested to be involved in microtubule dynamics, we have no clue of the mechanism by which spastin mutations may lead to degeneration of corticospinal axons. Therefore, we investigated the function of wild-type spastin and the molecular mechanism of neurodegeneration due to spastin mutations.1.Subcellular localization of wild-type and mutated spastinEach of overexpression of wild-type spastin or spastin^<A485V> construct showed a perinuclear localization in HeLa cells. A partial co-localization of spastin^<L426V> and and α-tubulin was observed with a filamentous pattern. Spastin^<TM+> construct localized to a perinuclear area, but spastin^<TM-> construct showed a diffuse intranuclear and cytoplasmic localization. In cells highly expressing spastin, the reduction in microtubule staining and broke … More n microtubules were observed. We found a nuclear export signal (NES) in spastin.2.Generation of polyclonal antibodies specific to spastinWe generated two polyclonal antibodies, sp-1 (amino acid residues 1-15) and sp-2 (recombinant amino acid residues 230-616), which are specific to spastin. Using these antibodies, we found that spastin showed an intranuclear localization in the interphase of HeLa cell-division cycles. In mitosis, spastin localized to the centrosomes in addition to the nuclei. Sapastin localized to the nuclei and the top of neurites in hNT2 cells.3.Analysis of the function of wild-type spastin using siRNAWhen the function of spastin was knocked down by siRNA, the cell division was disturbed in the anaphase of HeLa cell-division cycles. In hNT2 cells, the fragility of the top of neurites was observed. The gene profiling analysis was performed using two kinds of siRNA, and we found that the expression of M-Phase-Phosphoprotein 1(MPP1) is enhanced by knockdown of spastin function. Less

遗传性痉挛性截瘫的常染色体主导形式的大多数病例是由于SPG4基因中的突变所致，该基因编码Spastin，spastin是一种属于AAA家族的ATPase。尽管建议痉挛参与微管动力学，但我们尚无线索，氨氨饼突变可能导致皮质脊髓轴突变性的机制。 Therefore, we investigated the function of wild-type spastin and the molecular mechanism of neurodegeneration due to spastin mutations.1.Subcellular localization of wild-type and mutated spastinEach of overexpression of wild-type spastin or spastin^<A485V> construct shown a perinuclear localization in HeLa cells.用丝状图案观察到痉挛^<l426v>和α-微管蛋白的部分共定位。 Spastin^<tm+>构造位于核周区域的构造，但是Spastin^<tm->构建体显示了弥漫性内膜和细胞质定位。在高度表达痉挛的细胞中，观察到更多的N微管的微管染色和破裂。我们在Spastin中发现了一个核出口信号（NES）。与Spastinwe特异性的多克隆抗体的代代产生了两种多克隆抗体SP-1（氨基酸残基1-15）和SP-2（重组氨基酸残留230-616），这些抗体是特异性的。使用这些抗体，我们发现Spastin在HeLa细胞分割周期的相间中显示出内在的定位。在有丝分裂中，除了核外，Spastin还位于中心体。 sapastin位于Hnt2细胞中的核和神经的顶部。3.使用sirnawhen野生型翼蛋白的功能的分析，sirnawhen的功能被siRNA击倒，细胞分裂在Hela细胞分离循环的后期中扭曲。在Hnt2细胞中，观察到神经局部的脆弱性。使用两种siRNA进行了基因分析分析，我们发现通过敲除Spastin功能来增强M相磷蛋白1（MPP1）的表达。较少的