An analysis of molecular mechanism underlying spastin-induced spastic paraplegia and a strategy for the development of targeted therapies for the disease

常染色体显性遗传性痉挛性截瘫（HSPs）最常见的形式是由SPG4/SPAST基因突变引起的，该基因编码spastin，是atp酶AAA家族的一员。尽管spastin被认为与微管动力学有关，但我们还不清楚spastin突变导致皮质脊髓轴突变性的机制。因此，我们研究了痉挛蛋白的功能和痉挛蛋白突变引起的神经退行性变的分子机制。此外，我们试图寻找治疗SPG4的药物。(1)内源性spastin的亚细胞定位：我们利用spastin特异性抗体研究了内源性spastin在HeLa和NT2细胞中的亚细胞定位。在Hela细胞中，Spastin在间期主要定位于细胞核，在中期向中心体和纺锤体富集。在末期，spastin集中在细胞核和中间区，在中间区有强烈的染色。结果表明，spastin在细胞分裂微管切断过程中起重要作用。在NT2细胞中，spastin富集于生长锥和分支区。siRNA敲除spastin表达后，神经突伸长和肿胀异常，提示卵是远轴突生长所必需的。(2) SPG4可能治疗药物的筛选我们建立了SPG4可能治疗药物的筛选系统（NT2和SHSY5Y细胞中spastin表达siRNA敲除）。然后使用该系统筛选可能的SPG4治疗剂。长春花素虽然挽救了神经突的异常扩张，但也引起了神经元细胞的死亡。(3) sacsin基因表达谱我们进行了基因谱实验，鉴定了spastin低表达与siRNA敲低表达的基因。我们发现了一些可能与痉挛蛋白相关的候选基因。其中，一个基因与神经突的伸长和微管的稳定性有关，并与神经突顶部的spastin共定位。阐明与spastin相关的网络系统将有助于SPG4治疗方法的建立。少

The most common form of autosomal dominant hereditary spastic paraplegias (HSPs) is caused by mutations in the SPG4/SPAST gene, encoding spastin, a member of the AAA family of ATPases. Although spastin are suggested to be involved in microtubule dynamics, we have no due of the mechanism by which spastin mutations may lead to degeneration of corticospinal axons. Therefore, we investigated the function of spastin and the molecular mechanism underlying neurodegeneration due to spastin mutations. In addition, we attempted to find treatment agents for SPG4.(1) Subcellular localization of endogenous spastin :We investigated the subcellular localization of endogenous spastin in HeLa and NT2 cells using a spastin-specific antibody. In Hela cells, Spastin is predominantly localized in the nucleus during the interphase, with enrichment toward the centrosome and the spindle in the metaphase. In the telophase, spastin is concentrated in the nucleus and midzone region, with intense staining in the … More midbody. The results indicate that spastin plays an important role in cell division with microtubule severing process. In NT2 cells, spastin is enriched in the growth cone and in the branching regions. siRNA knock-down of spastin expression showed abnormal elongation and swelling of neurite, suggesting that spawn is essential far axon outgrouth.(2) Screening of possible treatment agents for SPG4We established a screening system (siRNA knock-down of spastin expression in NT2 and SHSY5Y cells) of possible treatment agents for SPG4. Then screening of possible treatment agents for SPG4 was performed using the system. Although vinblastin rescued the abnormal expansion of neurite, it also induced neuronal cell death.(3) Gene expression profiling on sacsinWe performed gene-profiling experiments to identify genes that are expressed at low levels together with siRNA knock-down of spastin expression. We found some candidate genes that could be associated with spastin. Among them, a gene was associated with the elongation of neurite and stability of microtubules, and was co-localized with spastin at the top of the neurite. To elucidate the net-work system associated with spastin will shed light on the establishment of treatment for SPG4. Less