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（HSP）的最常见形式是由SPG 4/SPAST基因突变引起的，SPG 4/SPAST基因编码痉挛蛋白，是ATP酶AAA家族的成员。虽然痉挛蛋白被认为参与微管动力学，我们没有由于痉挛蛋白突变可能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导致神经退行性变的分子机制。此外，我们试图找到SPG 4的治疗剂。(1)内源性spastin的亚细胞定位：我们使用spastin特异性抗体研究了HeLa和NT 2细胞中内源性spastin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， 关于我们 中体结果表明，spastin在细胞分裂过程中起着重要的作用。在NT 2细胞中，痉挛蛋白在生长锥和分支区域中富集。siRNA敲低spastin表达显示轴突异常伸长和肿胀，表明卵是轴突生长所必需的。(2)我们建立了SPG 4的可能治疗剂的筛选系统（NT 2和SHSY 5 Y细胞中spastin表达的siRNA敲低）。然后使用该系统进行SPG 4的可能治疗剂的筛选。虽然长春碱挽救了神经突起的异常扩张，但它也诱导了神经细胞死亡。(3)sacsin的基因表达谱分析我们进行了基因谱分析实验，以鉴定低水平表达的基因以及spastin表达的siRNA敲低。我们发现了一些可能与痉挛有关的候选基因。其中，一个基因与神经突的伸长和微管的稳定性相关，并与spastin共定位于神经突的顶端。阐明与spastin相关的网络系统将有助于SPG 4治疗的建立。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