The NIPA 1 protein in spastic paraplegia and development

描述（由申请人提供）：SPG 6是一种遗传性痉挛性截瘫，具有潜伏进行性下肢痉挛，伴有长中枢神经系统轴突变性。SPG 6基因定位于染色体15q11.2，我们在两个不相关的家族中发现了NIPA 1的显性负突变。NIPA 1和相邻的相关NIPA 2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，不连锁的家庭成员是突变的隐性鱼鳞病，皮肤病，这些研究牵连这个9-TM蛋白质家族的一个分支类花生生物学。NIPA 1在神经元和树突、内质网（ER）和相关囊泡中表达。在SPG 6中，我们提出，疾病的结果从预防的正常NIPA 1功能轴突维护和/或从二级机制，涉及未折叠蛋白反应（UPR）和ER捕获SPG蛋白。关于EGFP标记的Nipal和SPG 6的细胞系表达的初步数据支持这两种假设，因为Nipal诱导长的细胞延伸并且SPG 6突变诱导UPR。我们建议检查这些假设NIPA 1功能和SPG 6突变产生痉挛性截瘫的机制。目标1：为了检验我们的假设，即SPG 6突变产生UPR和/或以其他方式干扰NIPA 1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和神经元细胞中检查NIPA 1、突变体SPG 6和其他野生型SPG多肽的这些参数。目标二：为了确定Nipa 1在正常神经元中的作用，这是确定SPG 6突变是否干扰NIPA 1体内功能的先决条件，我们将（i）在组织培养中诱导和表征HeLa和神经元细胞延伸，（ii）在小鼠胚胎和出生后产生条件性Nipa 1功能丧失突变，以及（iii）通过吗啉代反义方法产生发育斑马鱼模型。目标3：为了检查SPG 6体内突变的分子病理学基础，我们将使用野生型和SPG 6转基因通过Nipa 1过表达产生痉挛性截瘫小鼠模型，检查与目的1和2相似的参数。目的4：为了检验SPG 6突变干扰NIPA 1膜转运的假设，将在非洲爪蟾卵母细胞中进行转运研究。我们的研究将建立NIPA 1的神经元作用，显性负突变产生神经系统疾病的机制，痉挛性截瘫中轴突神经变性的病理分子基础，并通过鉴定NIPA 1转运功能可能导致痉挛性截瘫和其他神经行为疾病的治疗靶点。了解神经元如何发育对于脊髓损伤和疾病的治疗至关重要。这项工作将使用遗传学和生物化学来确定一个对神经元功能和疾病重要的基因的功能。

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.