The NIPA 1 protein in spastic paraplegia and development

SPG6是一种遗传性痉挛截瘫，具有潜伏性进行性的下肢痉挛 中枢神经系统长轴突变性。SPG6基因座定位于染色体15q11.2， 在两个不相关的家系中发现NIPA1基因的显性-负性突变。NIPA1和相邻、相关 NIPA2基因编码9-跨膜(9-TM)结构域蛋白，我们推测其为转运蛋白。第三个， 无关联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，这与9- TM蛋白家族是二十烷类生物学的一个分支。NIPA1在神经元和树突中表达，在 内质网(ER)和相关的小泡。在SPG6中，我们提出疾病的原因是 在轴突维持和/或继发性机制中阻止正常的NIPA1功能 SPG蛋白的未折叠蛋白反应(UPR)和内质网捕获。细胞系的初步数据 绿色荧光蛋白标记的Nipal和SPG6的表达支持这两种假说，因为Nipal诱导长细胞 延伸和SPG6突变诱导UPR。我们建议检验NIPA1的这些假设 SPG6基因突变导致痉挛性截瘫的功能和机制。目标1：检查我们的 假设SPG6突变产生UPR和/或以其他方式干扰亚细胞拓扑 NIPA1的定位，或与其他SPG蛋白的相互作用，这些参数将被检测 NIPA1、突变型SPG6等野生型SPG多肽在HeLa和神经细胞中表达。目标2：确定 Nipal在正常神经元中的作用，这是确定SPG6突变是否干扰NIPA1的先决条件 在体内的功能，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乳头功能丧失突变，以及(Iii) 通过吗啡反义方法建立发育斑马鱼模型。目标3：检查 SPG6基因突变的分子病理学基础在体内，我们将建立痉挛性截瘫小鼠模型 通过野生型和SPG6转基因的Nipal过表达，检查与AIMS 1相似的参数 和2.目的4：验证SPG6突变干扰NIPA1膜转运的假设， 将在非洲爪哇卵母细胞中进行转运研究。我们的研究将确定神经细胞的作用 NIPA1，即显性-负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及NIPA1转运蛋白的鉴定 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 使用遗传学和生物化学来鉴定一种对神经元功能和疾病重要的Aene的功能。

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 Nipal 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 Nipal 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 Nipal 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 aenetics and biochemistry to identify the function of a aene important for neuron function and disease.