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

描述(由申请人提供)：轴突寻路是在发育过程中建立适当的神经元连接所必需的基本过程。阐明轴突生长和引导的机制不仅对了解神经系统建立的基本发育过程很重要，而且可能有助于揭示轴突连接缺陷引起的某些病理情况。事实上，伊丽莎白·恩格尔博士的实验室已经发现了几种神经疾病，这些疾病是由异常的神经连接引起的，被称为先天性颅神经功能障碍(CCDD)。在其中一种疾病，先天性眼外肌纤维化症3型(CFEOM3)中，患者由于眼外肌的脑神经支配失败而表现出眼球运动障碍。这种疾病背后的八个不同的错义突变被映射到β-微管蛋白亚型III(TUBB3)，这是一种高度动态的神经元特异性微管蛋白亚型，在轴突生长和寻路期间表达上调。每一种突变都会导致CFEOM3，但也可能伴随着额外的颅脑、脊髓和中央轴突的异常引导。一种携带人类CFEOM3患者中最常见突变并导致R262C氨基酸替换的敲入小鼠模型显示出轴突导向缺陷，而皮质结构没有改变，这表明这种疾病主要是由异常的轴突连接引起的。然而，TUBB3基因突变影响轴突微管和轴突导向的机制尚未被研究。这项训练包括两个目标，第一个目标是建立神经元模型系统，以研究TUBB3的病理性突变对轴突微管组织和动力学的影响。我将通过同源重组在小鼠胚胎干细胞中创造敲入病突变，然后将这些ES细胞分化为运动神经元。将通过免疫染色来分析生长锥中微管的定位和组织，并将通过生化方法和活细胞成像来研究轴突中微管的稳定性和动力学。第二个目标将利用ES细胞衍生的神经元模型系统来确定TUBB3突变对轴突引导的影响。具体地说，将研究生长锥体塌陷和轴突旋转对吸引和排斥的引导线索的反应。总而言之，这些目标的结果将有助于更好地理解微管在轴突引导中的作用，更重要的是，将阐明TUBB3突变导致人类神经疾病的机制。 与公共健康相关：在发育过程中，神经元延伸被称为轴突的长过程，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(TUBB3)的突变被证明通过影响微管细胞骨架并扰乱轴突对其适当突触靶点的引导而导致人类神经疾病。本研究旨在研究这些突变对微管组织和动力学的影响，以确定TUBB3在轴突引导中的发育作用，更重要的是，加深我们对由异常回路形成引起的人类神经发育障碍的理解。

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.