Hereditary Spastic Paraplegia due to SPG3A/atlastin mutation

描述（由申请人提供）：遗传性痉挛性截瘫（HSPs）是一种退行性脊髓疾病，可导致下肢痉挛性无力。最近，我们发现一个新的基因（SPGSA/atlastin）突变是常染色体显性SPG3A HSP的原因。这一进展已经转化为临床可用的热休克蛋白诊断测试，并为揭示热休克蛋白的分子病理生理学提供了机会。我们的建议建立在这一进展的基础上，并侧重于1)定义SPG3A HSP表型，并将临床变异与SPG3A突变和HSP修饰基因多态性联系起来；2)通过识别其相互作用因子了解SPGSA/atlastin的功能；3) SPG3A体外及动物模型的发病机制研究。特异性目的1将表征SPG3A HSP的表型并检查临床变异的基础。SPG3A表型可以是相当可变的，包括儿童和成人发病的无复杂性HSP和与低运动神经元体征相关的HSP。虽然临床变异有时与离散的SPGSA/atlastin突变相关，但具有相同突变的受试者之间的差异表明修饰因素的作用。已有良性热休克蛋白基因多态性修饰热休克蛋白表型的先例。因此，除了将SPG3A的HSP表型变异与离散的SPG3A突变联系起来外，Specific Aim 1还将评估SPGSA/atlastin、SPG4/spastin和其他HSP基因的良性多态性作为“候选修饰因子”。确定热休克蛋白的疾病修饰基因将扩大我们对热休克蛋白发病机制的认识。确定SPGSA/atlastin基因型-表型相关性将提示SPG3A功能域，并有助于我们研究与atlastin相互作用的因子。Specific Aim 2将通过识别其相互作用因子来深入了解SPGSA编码蛋白“atlastin”的功能。目前，关于atlasatin功能的线索主要来自3个方面：1)它定位于皮质运动神经元的顺式高尔基膜；2)与鸟苷酸结合蛋白1同源，鸟苷酸结合蛋白1是gtpase动力蛋白家族的成员，在各种核内体运输事件中发挥重要作用；3)其与HPK/ gck样激酶（HGK）的体外相互作用，HGK是c-Jun n末端激酶信号通路中的一种蛋白激酶。Specific Aim 2将鉴定和分析atlastin相互作用蛋白，研究已报道的atlastin- hgk相互作用，并确定热休克蛋白特异性SPGSA/atlastin突变是否会改变与atlastin相互作用的蛋白。特异性目的3将研究SPGSA/atlastin在体外和体内的发病机制。鉴于atlasatin在顺式高尔基体上的定位，我们特别感兴趣的是在体外和体内模型中检查高尔基体的结构和功能。我们将研究SPGSA/atlastin不足的培养神经元（通过RNAi方法创建）的形态学、分化和阿联素在细胞内的定位；野生型和突变型SPG3A cDNA过表达组。我们将研究已经在我们实验室创建的SPGSA/atlastin突变小鼠的行为和神经病理学。这些动物的初步分析揭示了年龄依赖性后肢运动障碍。通过鉴定SPG3A基因型-表型相关性，发现SPGSA/atlastin相互作用因子，揭示atlastin相互作用的代谢级联反应，并检查体外和体内HSP模型，本研究将为HSP和其他运动神经元疾病（包括肌萎缩性侧索硬化症）的病因和最终治疗提供见解。

DESCRIPTION (provided by applicant): The Hereditary Spastic Paraplegias (HSPs) are degenerative spinal cord disorders that cause disabling lower extremity spastic weakness. Recently, we discovered mutations in a novel gene (SPGSA/atlastin) as the cause of autosomal dominant SPG3A HSP. This advance has been translated into a clinically available diagnostic test for HSP and provides opportunities to expose HSP's molecular pathophysiology. Our proposal builds on this progress and focuses on 1) defining the SPG3A HSP phenotype and correlating clinical variatipn with SPG3A mutations and HSP modifying-gene polymorphisms; 2) understanding SPGSA/atlastin function by identifying its interacting factors; and 3) studying SPG3A pathogenesis in in vitro and animal models. Specific Aim 1 will characterize the phenotype and examine the basis of clinical variation in SPG3A HSP. The SPG3A phenotype can be quite variable and include childhood- and adult-onset uncomplicated HSP and HSP associated with lower motor neuron signs. Although clinical variation sometimes correlates with discrete SPGSA/atlastin mutation, variation between subjects with the same mutation indicates the action of modifying factors. There is precedent for benign HSP gene polymorphism to modify the HSP phenotype. Therefore, in addition to correlating SPG3A HSP phenotype variation with discrete SPG3A mutations, Specific Aim 1 will assess benign polymorphisms in SPGSA/atlastin, SPG4/spastin, and other HSP genes as "candidate modifying factors". Identifying disease modifying genes in HSP will expand our knowledge of HSP pathogenesis. Determining the SPGSA/atlastin genotype-phenotype correlations will suggest SPG3A functional domains and contribute to our studies of factors that interact with atlastin. Specific Aim 2 will provide insight into the function of SPGSA's encoded protein "atlastin" by identifying its interacting factors. Presently, clues to atlastin's function come from 3 sources: 1) its localization to cis-golgi membranes of cortical motor neurons; 2) it's homology to guanylate binding protein 1, a member of the dynamin family of GTPases that play essential roles in a wide variety of endosome trafficking events; and 3) its reported interaction in vitro with HPK/GCK-like kinase (HGK), a protein kinase in the c-Jun N-terminal kinase signaling (JNK) pathway. Specific Aim 2 will identify and analyze atlastin interacting proteins, investigate the reported atlastin-HGK interaction, and determine whether HSP-specific SPGSA/atlastin mutations change the proteins with which atlastin interacts. Specific Aim 3 will examine SPGSA/atlastin pathogenesis in vitro and in vivo. In view of atlastin's localization to cis-golgi, we are particularly interested in examining Golgi structure and function in in vitro and in vivo models. We will study morphology, differentiation, and atlastin intracellular location of cultured neurons bearing SPGSA/atlastin insufficiency (created through RNAi methods); and those with overexpression of wild-type and mutant SPG3A cDNA. We will study the behavior and neuropathology of SPGSA/atlastin mutation mice already created in our laboratory. Preliminary analysis of these animals reveals age-dependent hind limb motor impairment. By identifying SPG3A genotype-phenotype correlations, discovering SPGSA/atlastin-interacting factors, exposing metabolic cascades in which atlastin interacts, and examining in vitro and in vivo models of HSP, this investigation will provide insight into the causes and ultimately treatments for HSP and other motor neuron diseases including amyotrophic lateral sclerosis.