Hereditary Spastic Paraplegia due to SPG3A/atlastin mutation

描述(申请人提供)：遗传性痉挛性瘫痪(HSP)是一种退行性脊髓疾病，可导致肢体痉挛无力。最近，我们发现了一个新基因(SPGSA/atlastin)的突变，它是常染色体显性遗传性SPG3A HSP的原因。这一进展已经转化为临床上可用的HSP诊断试验，并为揭示HSP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，并集中在1)定义SPG3A HSP表型和临床变异与SPG3A突变和HSP修饰基因多态性的关系；2)通过确定其相互作用因素来了解SPGSA/atlastin的功能；以及3)研究SPG3A在体外和动物模型中的发病机制。具体目标1将描述SPG3A HSP的表型特征，并检查其临床变异的基础。SPG3A表型可以有很大的变化，包括儿童和成人起病的不复杂的HSP和与下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变异表明修饰因素的作用。良性HSP基因多态性改变HSP表型是有先例的。因此，除了将SPG3A热休克蛋白表型变异与离散的SPG3A突变相关联外，特殊目标1还将评估SPGSA/atlastin、SPG4/spastin和其他热休克蛋白基因的良性多态，将其作为“候选修饰因子”。识别HSP中的疾病修饰基因将扩大我们对HSP发病机制的认识。确定SPGSA/atlastin的基因型-表型相关性将提示SPG3A的功能域，并有助于我们对与atlastin相互作用的因素的研究。具体目标2将通过确定其相互作用因素，深入了解SPGSA编码的蛋白质“atlastin”的功能。目前，有关atlastin功能的线索来自三个来源：1)它定位于皮质运动神经元的顺式高尔基膜；2)它与鸟氨酸结合蛋白1同源，后者是GTP酶家族中的一个成员，在许多内体转运事件中发挥重要作用；3)它在体外与c-jun N末端信号通路中的一种蛋白激酶HPK/GCK样激酶(HGK)相互作用。具体目标2将鉴定和分析atlastin相互作用的蛋白，调查已报道的atlastin-HGK相互作用，并确定HSP特异性SPGSA/atlastin突变是否改变与atlastin相互作用的蛋白质。特异性目标3将在体外和体内检测SPGSA/atlastin的发病机制。鉴于阿特拉斯汀对顺式高尔基体的定位，我们特别感兴趣的是在体外和体内模型中检测高尔基体的结构和功能。我们将研究培养的SPGSA/atlastin缺乏的神经元(通过RNAi方法建立)以及野生型和突变型SPG3A基因过表达的神经元的形态、分化和细胞内定位。我们将研究我们实验室已经创造的SPGSA/atlastin突变小鼠的行为和神经病理学。对这些动物的初步分析显示，与年龄相关的后肢运动障碍。通过确定SPG3A基因-表型的相关性，发现SPGSA/atlastin相互作用的因素，揭示atlastin相互作用的代谢级联反应，以及研究HSP的体外和体内模型，这项研究将为HSP和其他运动神经元疾病(包括肌萎缩侧索硬化症)的病因和最终治疗提供洞察力。

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.