The NIPA 1 protein in spastic paraplegia and development

NIPA 1 蛋白在痉挛性截瘫和发育中的作用

• 批准号: 7032689
• 负责人: Robert D Nicholls
• 金额: $ 28.7万
• 依托单位: CHILDREN'S HOSP PITTSBURGH/UPMC HLTH SYS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-17 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7032689
• 关键词: axon; gene mutation; genes; paraplegia; proteins; role

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.

描述（由申请人提供）：SPG6，一种遗传性痉挛性截瘫，具有潜伏进行性下肢痉挛，伴有中枢神经系统长轴突退化。 SPG6 基因座映射到染色体 15q11.2，我们在两个不相关的家族中发现了 NIPA1 的显性失活突变。 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。第三位无血缘关系的家庭成员发生了隐性鱼鳞病（一种皮肤病）的突变；这些研究表明 9-TM 蛋白家族属于类二十烷酸生物学的一个分支。 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轴突维护中正常 NIPA1 功能的阻止和/或涉及 SPG 蛋白的未折叠蛋白反应 (UPR) 和 ER 捕获的二级机制引起的。 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 诱导长的细胞延伸，而 SPG6 突变诱导 UPR。我们建议检查 NIPA1 功能的这些假设以及 SPG6 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 SPG6 突变会产生 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，将在这些参数中检查 NIPA1、突变型 SPG6 和其他野生型 SPG 多肽。 HeLa 和神经元细胞。目标 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 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) 生成条件Nipa1 在小鼠胚胎和出生后发生功能丧失突变，以及 (iii) 通过吗啉代反义方法生成发育斑马鱼模型。目标 3：为了检查体内 SPG6 突变的分子病理学基础，我们将使用野生型和 SPG6 转基因通过 Nipa1 过表达来生成痉挛性截瘫小鼠模型，检查与目标 1 和 2 类似的参数。目标 4：检验 SPG6 的假设突变干扰 NIPA1 的膜运输，运输研究将在非洲爪蟾卵母细胞中进行。我们的研究将确定 NIPA1 的神经元作用、显性失活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，并通过识别 NIPA1 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。这项工作将利用遗传学和生物化学来识别对神经元功能和疾病很重要的基因的功能。