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: 7391079
• 负责人: Robert D Nicholls
• 金额: $ 27.87万
• 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 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7391079
• 关键词: 15q; Address; Adult; Affect; Age-Months; Amino Acids; Angelman Syndrome; Arachidonic Acids; Area; Axon; Behavioral; Biochemical; Biochemistry; Biological Assay; Biology; Birth; Cell Line; Cells; Chimeric Proteins; Chromosomes; Complementary DNA; Cultured Cells; Cytoplasm; Data; Dendrites; Development; Disease; Dominant-Negative Mutation; Eicosanoids; Electrodes; Electron Microscope; Electrophysiology (science); Embryo; Endoplasmic Reticulum; Endosomes; Family; Family member; Genes; Genomics; Hela Cells; Hereditary Spastic Paraplegia; Human; Ichthyoses; Immunohistochemistry; Individual; Induced Mutation; Knockout Mice; Lead; Life; Ligand Binding; Ligands; Localized; Location; Lower Extremity; Lysosomes; Maintenance; Maps; Membrane; Membrane Proteins; Modeling; Molecular; Motor; Mus; Mutate; Mutation; Nerve Degeneration; Neuraxis; Neurites; Neurologic; Neurons; Nuclear Envelope; Nucleotides; Pathway interactions; Patients; Phenotype; Play; Prevention; Protein Family; Protein Overexpression; Proteins; Purkinje Cells; Research Personnel; Role; Signal Transduction; Spastic Paraplegia; Spinal cord injury; Standards of Weights and Measures; Testing; Transcriptional Regulation; Transgenes; Transmembrane Domain; Transmembrane Transport; Transport Process; Vesicle; Western Blotting; Work; Xenopus oocyte; Zebrafish; base; in vivo; late endosome; loss of function mutation; member; mouse model; mutant; nervous system disorder; neurobehavioral; patch clamp; polypeptide; programs; receptor; response; skin disorder; solute; therapeutic target; tissue culture; voltage clamp

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.

SPG6是一种遗传性痉挛性截瘫，具有阴险的渐进性低超级痉挛。 长中枢神经系统轴突的变性。 SPG6基因座图到15q11.2染色体，我们 确定了两个无关家族的NIPA1中的显性阴性突变。 NIPA1和相关的 NIPA2基因编码9跨膜（9-TM）结构蛋白，我们假设是转运蛋白。第三， 未连接的家庭成员在隐性鱼质病，皮肤病中突变。这些研究暗示了这9- eicosanoid生物学的一个分支中的TM蛋白家族。 NIPA1在神经元和树突中表达 内质网（ER）和相关囊泡。在SPG6中，我们提出疾病是由 预防轴突维护中的正常NIPA1功能和/或涉及辅助机制 SPG蛋白的展开蛋白质反应（UPR）和ER捕获。细胞系的初步数据 EGFP标记的NIPAL和SPG6的表达支持这两个假设，因为NIPAL诱导了长细胞 扩展和SPG6突变诱导UPR。我们建议检查NIPA1的这些假设 SPG6突变产生痉挛性截瘫的功能和机制。目标1：检查我们的 SPG6突变会产生UPR和/或以其他方式干扰拓扑，亚细胞的假设 NIPA1的定位，或与其他SPG蛋白的相互作用，将检查这些参数 HELA和神经元细胞中的NIPA1，突变体SPG6和其他野生型SPG多肽。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 在体内功能，我们将（i）诱导和表征组织培养中的HeLa和神经元细胞延伸，（ii） 在小鼠胚胎和产后产生有条件的nipal功能突变，以及（iii） 通过形态反义方法生成发育的斑马鱼模型。目标3：检查 SPG6突变在体内的分子病理基础，我们将生成痉挛性截瘫小鼠模型 通过使用WildType和Spg6转基因的NIPAL过表达，检查与目标1相似的参数 和2。目标4：检验一个假设，即SPG6突变干扰NIPA1的膜转运， 运输研究将在爪蟾卵母细胞中进行。我们的研究将确定 NIPA1，主导阴性突变产生神经系统疾病的机制， 痉挛性截瘫的轴突神经变性的病原体基础，并通过鉴定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标。 了解神经元如何发展对于治疗脊髓损伤和疾病至关重要。这项工作将 使用配合学和生物化学来识别AENE对神经元功能和疾病重要的功能。