The effect of pathological mutations in beta-III tubulin on microtubules and axon

β-III微管蛋白病理突变对微管和轴突的影响

• 批准号: 8313434
• 负责人: Adrianne Lynn Kolpak
• 金额: $ 4.92万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-03-01 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8313434
• 关键词: Actins; Affect; Amino Acid Substitution; Architecture; Axon; Biochemical; Biological Assay; Biological Models; Cephalic; Congenital Disorders; Cranial Nerves; Cues; Cytoskeleton; Data; Defect; Development; Developmental Process; Disease; Distal; Ensure; Environment; Etiology; Exhibits; Fellowship; Fibrosis; Genes; Genetic; Goals; Growth; Growth Cones; Human; Knock-in Mouse; Lead; Length; Life; Light; Maps; Mediating; Mental Retardation; Microfilaments; Microscopy; Microtubules; Missense Mutation; Motor Neurons; Mus; Mutation; Nerve; Nervous System Physiology; Nervous system structure; Neurodevelopmental Disorder; Neurons; Ocular Motility Disorders; Pathology; Patients; Peripheral; Phenotype; Physiological; Play; Plus End of the Microtubule; Polymers; Polyneuropathy; Process; Protein Binding; Protein Isoforms; Reporting; Role; Signal Transduction; Spinal; Synapses; Syndrome; Testing; Time; Training; Tubulin; axon growth; axon guidance; base; beta Tubulin; cell motility; cellular imaging; embryonic stem cell; extracellular; homologous recombination; insight; migration; mouse model; mutant; nerve supply; nervous system disorder; neuron development; novel; oculomotor; orbit muscle; overexpression; response

DESCRIPTION (provided by applicant): Axon pathfinding is an essential process required for the establishment of proper neuronal connections during development. Elucidating the mechanisms governing axon growth and guidance will be important not only for understanding the basic developmental processes by which the nervous system is established but may also shed light on certain pathological conditions arising from axon wiring defects. In fact, Dr. Elizabeth Engle's lab has identified several neurological disorders resulting from aberrant nerve connectivity, known as the congenital cranial dysinnervation disorders (CCDDs). In one of these disorders, congenital fibrosis of the extraocular muscles type 3 (CFEOM3), patients exhibit ocular motility defects due to failed cranial nerve innervation of the extraocular muscles. Eight different missense mutations underlying this disorder mapped to beta-tubulin isotype III (TUBB3), a highly dynamic, neuron-specific tubulin isoform whose expression is upregulated during the period of axon growth and pathfinding. Each mutation results in CFEOM3, but can also be accompanied by aberrant guidance of additional cranial, spinal, and central axons. A knock-in mouse model harboring the most common mutation identified in human CFEOM3 patients, and resulting in the R262C amino acid substitution, exhibits axon guidance defects without alterations in cortical architecture, suggesting this disease is primarily caused by aberrant axon connectivity. However, the mechanisms by which mutations in TUBB3 affect axonal microtubules and axon guidance have not yet been investigated. This training fellowship consists of two aims, the first of which proposes to establish a neuronal model system in order to investigate the effect of pathological mutations in TUBB3 on microtubule organization and dynamics in axons. I will create knock-in disease mutations in mouse embryonic stem (ES) cells through homologous recombination and then differentiate these ES cells into motor neurons. The localization and organization of microtubules in the growth cone will be analyzed by immunostaining and the stability and dynamics of microtubules in the axons will be investigated through a biochemical approach and live cell imaging. The second aim will utilize the ES cell-derived neuronal model system in order to determine the effect of the TUBB3 mutations on axon guidance. Specifically, growth cone collapse and axon turning in response to attractive and repulsive guidance cues will be investigated. Collectively, the results from these aims will lead t a better understanding of the role of microtubules in axon guidance, and, more importantly, will elucidate the mechanisms by which TUBB3 mutations result in human neurological disorders. PUBLIC HEALTH RELEVANCE: During development, neurons extend long processes known as axons, which are required to form precise connections to ensure proper nervous system function. Recently, mutations in the beta-III tubulin gene (TUBB3) have been shown to cause human neurological disorders by affecting the microtubule cytoskeleton and perturbing the guidance of axons to their appropriate synaptic targets. This proposal seeks to investigate the effects of these mutations on microtubule organization and dynamics in order to identify the developmental role of TUBB3 in axon guidance and, more importantly, to increase our understanding of human neurodevelopmental disorders resulting from abnormal circuit formation.

描述（由申请方提供）：轴突寻路是发育期间建立适当神经元连接所需的重要过程。阐明轴突生长和引导的机制不仅对于理解建立神经系统的基本发育过程非常重要，而且还可能揭示轴突布线缺陷引起的某些病理状况。事实上，伊丽莎白·恩格尔博士的实验室已经确定了几种由异常神经连接引起的神经系统疾病，称为先天性颅神经支配障碍（CCDD）。在这些病症之一中，先天性眼外肌纤维化3型（CFEOM 3），患者由于眼外肌的颅神经神经支配失败而表现出眼运动缺陷。八种不同的错义突变导致这种疾病映射到β-微管蛋白同种型III（TUBB 3），这是一种高度动态的神经元特异性微管蛋白同种型，其表达在轴突生长和寻路期间上调。每个突变都会导致CFEOM 3，但也可能伴随着额外的颅，脊髓和中央轴突的异常指导。携带在人类CFEOM 3患者中鉴定的最常见突变并导致R262 C氨基酸取代的敲入小鼠模型表现出轴突引导缺陷而没有皮质结构的改变，表明这种疾病主要是由异常轴突连接引起的。然而，TUBB 3突变影响轴突微管和轴突导向的机制尚未研究。该培训奖学金由两个目标组成，第一个目标是建立一个神经元模型系统，以研究TUBB 3的病理突变对微管组织和轴突动力学的影响。我将通过同源重组在小鼠胚胎干细胞（ES）中制造敲入疾病突变，然后将这些ES细胞分化为运动神经元。生长锥中微管的定位和组织将通过免疫染色进行分析，轴突中微管的稳定性和动力学将通过生物化学方法和活细胞成像进行研究。第二个目标将利用ES细胞衍生的神经元模型系统，以确定TUBB 3突变对轴突导向的影响。具体而言，生长锥崩溃和轴突转向的吸引力和排斥性的指导线索将进行调查。总的来说，这些目标的结果将使我们更好地理解微管在轴突导向中的作用，更重要的是，将阐明TUBB 3突变导致人类神经系统疾病的机制。 公共卫生相关性：在发育过程中，神经元延伸出被称为轴突的长过程，这些轴突是形成精确连接以确保神经系统正常功能所必需的。最近，β-III微管蛋白基因（TUBB 3）的突变已被证明会通过影响微管细胞骨架和扰乱轴突对其适当突触靶点的引导而引起人类神经系统疾病。该提案旨在研究这些突变对微管组织和动力学的影响，以确定TUBB 3在轴突指导中的发育作用，更重要的是，增加我们对异常回路形成导致的人类神经发育障碍的理解。