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

大多数常染色体显性遗传性痉挛性截瘫的病例是由于SPG4基因的突变所致，SPG4基因编码属于AAA家族的一种ATPase-spastin。虽然spastin被认为与微管动力学有关，但我们还不知道spastin突变导致皮质脊髓轴突退化的机制。因此，我们研究了野生型spastin的功能及其引起神经变性的分子机制。1.野生型和突变型spastin的亚细胞定位野生型spastin或spastin^&lt；A485V&gt；构建的每一个过表达的spastin都显示在HeLa细胞的核周定位。Spastin^&lt；L426V&gt；和α-Tubuin的部分共定位呈丝状。Spastin^&lt；TM&gt；结构定位于核周，而Spastin^&lt；TM-&gt；结构呈弥漫性核内和胞浆定位。在高表达spastin的细胞中，微管染色减少并断裂…可见较多的n微管。2.针对Spastin的多克隆抗体的制备我们制备了两种针对Spastin的多克隆抗体SP-1(氨基酸残基1-15)和SP-2(重组氨基酸残基230-616)。利用这些抗体，我们发现spastin在HeLa细胞分裂周期的间期显示出核内定位。在有丝分裂中，除细胞核外，spastin还定位于中心体。Sapastin定位于hNT2细胞的核和突起顶端。3.siRNA分析野生型spastin的功能当siRNA下调spastin的功能时，细胞分裂在HeLa细胞分裂周期的后期受到干扰。在hNT2细胞中，观察到突起顶端的脆性。利用两种siRNA进行基因表达谱分析，我们发现M相磷酸蛋白1(MPP1)的表达通过下调spastin功能而增强。较少

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