Novel mechanistic study of CMT2D neuropathy

CMT2D 神经病的新机制研究

基本信息

批准号：
8656827
负责人：
SAMUEL L. PFAFF
金额：
$ 24.01万
依托单位：
SALK INSTITUTE FOR BIOLOGICAL STUDIES
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-05-01 至 2015-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8656827
关键词：
1-Phosphatidylinositol 3-Kinase Affect Afferent Neurons Agonist Amino Acids Amino Acyl Transfer RNA Amino Acyl-tRNA Synthetases Animal Model Axon Behavioral Research Binding Biochemical Biological Biological Assay Biomedical Research Cell Line Charcot-Marie-Tooth Disease Clinical Research Collaborations Cytoplasm Cytoplasmic Protein Data Development Disease Distal Enzyme Gene Enzymes Equilibrium Ethylnitrosourea Face Family Future Gene Mutation Gene Proteins Genes Genetic Glycine Goals Grant Growth Cones Hand Hereditary Motor and Sensory Neuropathies Housekeeping In Vitro Investigation Lead Ligands Ligase Measures Morphology Motor Motor Neurons Mus Muscle Muscle Weakness Mutant Strains Mice Mutation Neurons Neuropathy Neuropilins Pathway interactions Peripheral Peripheral Nervous System Diseases Phenotype Phylogenetic Analysis Point Mutation Protein Isoforms Protein Secretion Proteins RNA Research Research Support Role Sensory Serum Signal Transduction Spinal Cord Staging Testing Therapeutic Agents Translations Vascular Endothelial Growth Factors Western Blotting Yang axon growth axonal degeneration base clinically relevant extracellular gain of function gain of function mutation gene function glycine-tRNA hereditary peripheral nervous system disorder high reward high risk hindbrain in vivo migration motor deficit motor neuron development mouse model mutant neuron development neuron loss neuronal cell body neurotrophic factor novel prevent protein function receptor receptor binding vector

项目摘要

Charcot Marie Tooth 2D (CMT2D) is caused by dominant mutations in the glycine RNA synthetase gene (GlyRS). Despite the ubiquitous requirement for this cytoplasmic enzyme in generating glycine-tRNA for translation, mutations cause selective peripheral neuron axon degeneration leading to motor and sensory deficits. Although CMT2D was initially thought to be caused by inadequate GlyRS enzyme, previous genetic studies indicate that CMT2D is caused by a neomorphic function of GlyRS (i.e. gain-of-function). The mechanism of action by which mutant GlyRS causes CMT2D is not known and therefore is the focus of this grant. Preliminary in vitro and biochemical data from our collaborator Xiang-Lei Yang's lab has unmasked a heretofore unknown biological pathway in which GlyRS is secreted and binds competitively with VEGF to the Nrp1 receptor. Since motor neurons express Nrp1, and VEGF isoform 164 has neurotrophin-activity, they hypothesize that the novel extracellular interaction between mutant GlyRS and Nrp1 underpins CMT2D. The goal in this exploratory R21 grant is to establish whether key aspects of their hypothesis are correct in order to determine if VEGF can be used as a therapeutic agent for CMT2D disease. The four aims are (1) to establish whether GlyRS (normal and mutant protein) is secreted in mice, (2) to characterize the motor deficits in CMT2D mice and determine if GlyRS genetically interacts with VEGF and Nrp1 in vivo, (3) to assay the influence of GlyRS on Nrp1 signaling, and (4) to express VEGF in CMT2D mouse muscles using AAV-VEGF vectors and assay whether the progression of peripheral neuropathy (i.e. axon loss) is slowed or prevented. These studies may lead to a paradigm shift in our understanding of normal GlyRS function as an extracellular ligand in a broad array of biological contexts, but the main goal here is to determine whether mutant GlyRS has acquired a neomorphic activity as a VEGF antagonist of Nrp1 signaling in motor neurons. This grant aims to establish sufficient preliminary data to justify further investigation of VEGF as a feasible and novel treatment for CMT2D.

Charcot Marie Tooth 2D (CMT2D) 是由甘氨酸 RNA 合成酶基因 (GlyRS) 显性突变引起的。尽管在生成甘氨酸-tRNA 进行翻译时普遍需要这种细胞质酶，但突变会导致选择性周围神经元轴突变性，从而导致运动和感觉缺陷。尽管 CMT2D 最初被认为是由 GlyRS 酶不足引起的，但之前的遗传学研究表明，CMT2D 是由 GlyRS 的新形态功能（即功能获得性）引起的。突变型 GlyRS 导致 CMT2D 的作用机制尚不清楚，因此是本次资助的重点。我们的合作者Xiang-Lei Yang实验室的初步体外和生化数据揭示了一种迄今为止未知的生物途径，其中GlyRS被分泌并与VEGF竞争性地结合到Nrp1受体上。由于运动神经元表达 Nrp1，并且 VEGF 同工型 164 具有神经营养蛋白活性，因此他们推测突变型 GlyRS 和 Nrp1 之间的新型细胞外相互作用是 CMT2D 的基础。这项探索性 R21 资助的目标是确定他们的假设的关键方面是否正确，以确定 VEGF 是否可以用作 CMT2D 疾病的治疗剂。这四个目标是 (1) 确定 GlyRS（正常蛋白和突变蛋白）是否在小鼠体内分泌，(2) 表征 CMT2D 小鼠的运动缺陷并确定 GlyRS 是否在体内与 VEGF 和 Nrp1 存在遗传相互作用，(3)测定 GlyRS 对 Nrp1 信号传导的影响，以及（4）使用 AAV-VEGF 载体在 CMT2D 小鼠肌肉中表达 VEGF，并测定周围神经病变（即轴突）是否进展损失）被减缓或防止。这些研究可能会导致我们对正常 GlyRS 在广泛的生物学背景下作为细胞外配体的功能的理解发生范式转变，但这里的主要目标是确定突变型 GlyRS 是否已获得作为 Nrp1 信号传导的 VEGF 拮抗剂的新形态活性在运动神经元中。该资助旨在建立足够的初步数据，以证明 VEGF 作为 CMT2D 可行且新颖的治疗方法的进一步研究是合理的。