Mechanisms of SPG4 Hereditary Spastic Paraplegia

项目摘要/摘要 遗传性痉挛性瘫痪(HSP)是遗传性神经退行性疾病，在这种疾病中， 皮质脊髓轴索变性导致肢体无力、痉挛和步态缺陷。这些 症状是由皮质脊髓轴突变性的垂死模式引起的，也表现为显著的 病理意义不明的肿胀。最常见的HSP形式，称为SPG4-HSP，由以下原因引起 Spast基因的突变，该基因编码一种名为spastin的微管切断蛋白。到目前为止， SPG4-HSP病因学的流行机制假说是单倍体不足，这意味着 皮质脊髓束因功能性痉挛不足而退化。然而，几种主要疾病 这种病因学并不能很好地解释这些特征，也不清楚微管切断如何减少。 促进皮质脊髓轴突变性。提供新颖的信息，可能会填补我们在 对SPG4-HSP致病机制的了解，主要研究人员最近的工作揭示了SPG4-HSP的毒性特性 突变的spastin蛋白，表明在SPG4-HSP中存在功能增强机制。奇怪的是，两者 机制对快速轴突运输(FAT)产生负面影响，这是一种由分子马达蛋白驱动的细胞过程 这使得囊泡的货物可以沿着轴突双向移动。基于一个强有力的实验前提， 在这个多PI赠款方案中假设微管组织中的异常与 Spastin水平降低会导致脂肪不足和轴突肿胀(功能丧失)。另一方面，有毒的影响 突变的spastin蛋白导致不同的脂肪缺陷，这些脂肪缺陷是由酪蛋白激酶2(CK2)介导的 缺陷会促进皮质脊髓轴突退化(功能获得)。前者使轴突更加脆弱， 但对于皮质脊髓轴突变性来说，后者才是足够的。拟议的工作旨在测试这些 通过直接比较带有单一spast等位基因(spast/-)的小鼠模型与转基因小鼠的假设 具有两个内源性小鼠spast等位基因的模型，该模型额外表达携带A基因的人spastin 与SPG4-HSP(spastin-C448Y小鼠)相关的致病突变。在目标1中，这些模型将是 分别与在皮质脊髓运动神经元(CSMN)选择性表达EGFP的小鼠杂交，以便 对疾病的功能丧失和功能恢复的贡献可以进行调查。在目标2中，将研究脂肪不足。 在从这些动物培养的神经元中，将测试关于缺陷病因的具体假设。在……里面 目的3.利用转基因spastin-C448Y小鼠进行自噬实验研究 通过实验提高或降低CK2水平，以检验这些操作将防止 或减少皮质脊髓轴突变性和相关的行为缺陷。这件事的总体意义 该项目旨在建立SPG4-HSP的潜在机制，并开辟一条有效的治疗途径 病人。

PROJECT SUMMARY / ABSTRACT Hereditary Spastic Paraplegias (HSP) are heritable neurodegenerative diseases in which progressive degeneration of corticospinal axonal tracts results in limb weakness, spasticity and gait deficiencies. These symptoms result from a dying back pattern of degeneration of corticospinal axons, which also display prominent swellings of unclear pathological significance. The commonest form of HSP, termed SPG4-HSP, is caused by mutations in the SPAST gene, which codes for a microtubule-severing protein called spastin. To date, the prevailing mechanistic hypothesis for the etiology of SPG4-HSP is haploinsufficiency, meaning that the corticospinal tracts degenerate because of insufficient functional spastin. However, several major disease features are not readily explained by this etiology, and it is not clear how reduced microtubule severing would promote corticospinal axonal degeneration. Providing novel information that may fill a major gap in our knowledge of SPG4-HSP pathogenesis, recent work of the Principal Investigators revealed toxic properties of mutant spastin proteins, suggesting that a gain-of-function mechanism operates in SPG4-HSP. Curiously, both mechanisms negatively affect fast axonal transport (FAT), a cellular process fueled by molecular motor proteins that allows bidirectional movement of vesicular cargoes along axons. Based on a strong experimental premise, it is hypothesized in this multi-PI grant proposal that abnormalities in microtubule organization associated with reduced spastin levels cause FAT deficits and axonal swellings (loss-of-function). On the other hand, toxic effects of mutant spastin protein cause different FAT deficits that are mediated by casein kinase 2 (CK2), and these deficits promote corticospinal axon degeneration (gain-of-function). The former makes the axon more vulnerable, but it is the latter that suffices for corticospinal axon degeneration. The proposed work seeks to test these hypotheses by directly comparing a mouse model with a single SPAST allele (SPAST +/-) with a transgenic mouse model with both endogenous mouse SPAST alleles intact that additionally expresses human spastin bearing a pathogenic mutation associated with SPG4-HSP (spastin-C448Y mice). In Aim 1, these models will be individually crossed with mice that selectively express eGFP in corticospinal motor neurons (CSMN), so that loss-of and gain-of-function contributions to the disease can be investigated. In Aim 2, FAT deficits will be studied in neurons cultured from these animals, and specific hypotheses for the etiology of the deficits will be tested. In Aim 3, studies are proposed using transgenic spastin-C448Y mice in which autophagy is experimentally enhanced or CK2 levels are experimentally reduced, to test the hypothesis that these manipulations will prevent or reduce corticospinal axon degeneration and associated behavioral deficits. The overall significance of this project is to establish mechanisms underlying SPG4-HSP and forge a path toward effective therapies for patients.