The NIPA 1 protein in spastic paraplegia and development

描述（申请人提供）：SPG6，遗传性痉挛性截瘫，隐蔽性进行性下肢痉挛伴长中枢神经系统轴突变性。SPG6位点定位于染色体15q11.2，我们在两个不相关的家族中发现了NIPA1的显性阴性突变。NIPA1和邻近的相关NIPA2基因编码9-跨膜（9-TM）结构域蛋白，我们假设它们是转运蛋白。第三个无血缘关系的家庭成员在隐性鱼鳞病（一种皮肤病）中发生突变；这些研究表明9-TM蛋白家族是类二十烷类生物的一个分支。NIPA1在神经元和树突、内质网（ER）和相关囊泡中表达。在SPG6中，我们提出疾病是由于NIPA1在轴突维持中的正常功能被阻止和/或SPG蛋白的未折叠蛋白反应（UPR）和内质网捕获的次要机制引起的。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诱导长细胞延伸，而SPG6突变诱导UPR。我们建议研究这些关于NIPA1功能的假设以及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突变产生UPR和/或以其他方式干扰NIPA1的拓扑结构、亚细胞定位或与其他SPG蛋白的相互作用，这些参数将在HeLa和神经元细胞中检测NIPA1、突变SPG6和其他野生型SPG多肽。目的2：为了确定Nipa1在正常神经元中的作用，这是确定SPG6突变是否干扰Nipa1在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和神经元细胞扩展，（ii）在小鼠胚胎和出生后产生条件Nipa1功能丧失突变，以及（iii）通过morpholino反义方法生成发育中的斑马鱼模型。目的3：为了研究SPG6体内突变的分子病理基础，我们将使用野生型和SPG6转基因通过Nipa1过表达产生痉挛性截瘫小鼠模型，检测与目的1和目的2相似的参数。目的4：为了验证SPG6突变干扰NIPA1膜运输的假设，将在非洲爪蟾卵母细胞中进行运输研究。我们的研究将确定NIPA1的神经元作用，显性负突变产生神经系统疾病的机制，痉挛性截瘫轴突神经变性的病理分子基础，并通过确定NIPA1的转运功能可能找到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.