The NIPA 1 protein in spastic paraplegia and development

描述(申请人提供)：SPG6是一种遗传性痉挛截瘫，具有潜伏的进行性下肢痉挛，伴有中枢神经系统长轴突的变性。SPG6基因定位于染色体15q11.2，我们在两个不相关的家系中发现了NIPA1的显性-负性突变。NIPA1和邻近的相关NIPA2基因编码9-跨膜(9-TM)结构域蛋白，我们假设是转运蛋白。第三个无连锁的家庭成员在隐性鱼鳞病中发生突变，这是一种皮肤病；这些研究表明，这个9-TM蛋白家族与二十烷类生物学的一个分支有关。NIPA1表达于神经元和树突、内质网(ER)和相关的小泡。在SPG6中，我们认为疾病是由于轴突维持中正常的NIPA1功能的阻止和/或涉及SPG蛋白的未折叠蛋白反应(UPR)和内质网捕获的二级机制造成的。关于EGFP标记的Nipal和SPG6细胞系表达的初步数据支持这两种假设，因为Nipal诱导细胞延长，SPG6突变诱导UPR。我们建议检验NIPA1功能的这些假说以及SPG6突变导致痉挛性截瘫的机制。目的1：为了验证我们的假设，即SPG6突变会产生UPR和/或干扰NIPA1的拓扑结构、亚细胞定位或与其他SPG蛋白的相互作用，将在HeLa和神经细胞中检测NIPA1、突变体SPG6和其他野生型SPG多肽的这些参数。目的：为了确定Nipa1在正常神经元中的作用，这是确定SPG6突变是否在体内干扰NIPA1功能的前提，我们将(I)在组织培养中诱导和鉴定HeLa和神经细胞延伸，(Ii)在小鼠胚胎和出生后产生条件性Nipa1功能丧失突变，(Iii)通过吗啡反义方法建立发育斑马鱼模型。目的3：为了探讨SPG6突变在体内的分子病理学基础，我们将利用野生型和SPG6转基因基因通过Nipa1的过度表达建立痉挛截瘫小鼠模型，并检验与AIMS 1和2相似的参数。目的4：为了验证SPG6突变干扰NIPA1膜转运的假说，我们将在非洲爪哇卵母细胞中进行转运研究。我们的研究将确定NIPA1的神经元作用，显性-阴性突变导致神经疾病的机制，痉挛截瘫轴突神经变性的病理分子基础，并通过确定NIPA1的运输功能可能导致痉挛截瘫和其他神经行为疾病的治疗靶点。了解神经元是如何发育的对于脊髓损伤和疾病的治疗至关重要。这项工作将利用遗传学和生物化学来鉴定对神经元功能和疾病至关重要的基因的功能。

DESCRIPTION (provided by applicant): SPG6, a hereditary spastic paraplegia, has insidiously progressive lower-extremity spasticity with degeneration of long central nervous system axons. The SPG6 locus maps to chromosome 15q11.2 and we identified dominant-negative mutations in NIPA1 in two unrelated families. NIPA1 and the adjacent, related NIPA2 gene encode 9-transmembrane (9-TM) domain proteins we hypothesize are transporters. A third, unlinked family member is mutated in a recessive ichthyosis, a skin disease; these studies implicate this 9-TM protein family in one branch of eicosanoid biology. NIPA1 is expressed in neurons and dendrites, in the endoplasmic reticulum (ER) and associated vesicles. In SPG6, we propose that disease results from prevention of a normal NIPA1 function in axonal maintenance and/or from a secondary mechanism involving an unfolded protein response (UPR) and ER trapping of SPG proteins. Preliminary data on cell line expression of EGFP-tagged Nipal and SPG6 supports both hypotheses, since Nipal induces long cellular extensions and the SPG6 mutation induces the UPR. We propose to examine these hypotheses of NIPA1 function and the mechanism by which SPG6 mutations produce spastic paraplegia. Aim 1: To examine our hypothesis that SPG6 mutations generate an UPR and/or otherwise interfere with topology, subcellular localization of NIPA1, or of interactions with other SPG proteins, these parameters will be examined for NIPA1, mutant SPG6 and other wildtype SPG polypeptides in HeLa and neuronal cells. Aim 2: To determine the role of Nipa1 in normal neurons, a prerequisite to determining if SPG6 mutations interfere with NIPA1 function in vivo, we will (i) induce and characterize HeLa and neuronal cell extensions in tissue culture, (ii) generate a conditional Nipa1 loss of function mutation in the mouse embryo and postnatally, and (iii) generate developmental zebrafish models by a morpholino antisense approach. Aim 3: To examine the molecular pathological basis for SPG6 mutations in vivo, we will generate spastic paraplegia mouse models by Nipa1 overexpression using wildtype and SPG6 transgenes, examining similar parameters as for Aims 1 and 2. Aim 4: To test a hypothesis that SPG6 mutations interfere with membrane transport by NIPA1, transport studies will be performed in Xenopus oocytes. Our studies will establish the neuronal roles of NIPA1, the mechanisms by which dominant-negative mutations produce neurological disease, the pathomolecular basis of axonal neurodegeneration in spastic paraplegia, and by identifying NIPA1 transport functions may lead to therapeutic targets in spastic paraplegia and other neurobehavioral diseases. Understanding how neurons develop is critical to therapy for spinal cord injury and disease. This work will use genetics and biochemistry to identify the function of a gene important for neuron function and disease.