Genetic analyses of axon transport and microtubule dynamics in Zebrafish

斑马鱼轴突运输和微管动力学的遗传分析

• 批准号: 8873990
• 负责人: Alex Nechiporuk
• 金额: $ 31.16万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-25 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8873990
• 关键词: Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Animals; Axon; Axonal Transport; Biological Assay; Biological Models; Candidate Disease Gene; Carrier Proteins; Cells; Charcot-Marie-Tooth Disease; Communication; Communities; Defect; Development; Disease; Eligibility Determination; Embryo; Embryonic Development; Ensure; Etiology; Exhibits; Failure; Family; Foundations; Functional disorder; Genes; Genetic; Genetic Screening; Goals; Health; Home environment; Image; Imagery; Institution; Intracellular Transport; Investigation; Laboratories; Larva; Lead; Lesion; Letters; Life; Link; Location; Maintenance; Maps; Mediating; Microtubules; Molecular; Motor; Mutagenesis; Mutate; Mutation; Nerve; Nerve Degeneration; Nervous system structure; Neurodegenerative Disorders; Neurodevelopmental Disorder; Neurons; Organelles; Orthologous Gene; Pattern; Phenotype; Play; Process; Proteins; Protocols documentation; Reagent; Regulation; Role; Siblings; Signal Transduction; Staging; Swelling; Synapses; System; Techniques; Testing; Tissues; Transcript; Transport Process; Vertebrates; Zebrafish; axonal degeneration; base; effective therapy; genetic analysis; genetic linkage analysis; in vivo; in vivo Model; innovation; mutant; myelination; nervous system development; neurodevelopment; neuronal cell body; novel; positional cloning; premature; public health relevance; screening; stem; tool; zebrafish genome

DESCRIPTION (provided by applicant): The coordination of intracellular protein/organelle transport and microtubule dynamics plays an essential role in axon outgrowth and circuit formation during nervous system development and maintenance. Abnormalities in these processes are associated with a number of neurodevelopmental and neurodegenerative disorders, such as Charcot-Marie-Tooth disease, Alzheimer's disease, and Amyotrophic Lateral Sclerosis. Despite the pervasiveness of these disorders, there are no effective treatments. This deficiency can be partially attributed to the lack of an in vivo vertebrate system, which is needed to effectively analyze how alterations in axon transport or microtubule dynamics lead to failed axon extension or axonal degeneration. Our long-term goal is to define the genetic basis of the cellular and molecular mechanisms, which regulate axon transport and microtubule dynamics using the zebrafish as an in vivo model system. The overall objective of this proposal is to use forward genetic screening to find factors that regulate axon transport and microtubule dynamics during development of the vertebrate nervous system. Our preliminary studies confirm that this screen is feasible. We conducted a pilot screen that yielded 5 mutant strains exhibiting phenotypes consistent with defective axon transport and/or disrupted microtubule dynamics including: axon truncation, nerve degeneration and axonal swellings. We have also developed a number of assays to characterize microtubule-motor mediated transport and cytoskeletal dynamics in our mutant strains, including imaging approaches to visualize these processes in intact zebrafish embryos and larvae. Using these approaches, we have demonstrated that one of the mutants (llg1), which contains a mutation in an ortholog of Jnk-interacting protein 3 (Jip3) has specific defects in axon transport and simultaneously exhibits abnormal microtubule dynamics. In Aim 1, we propose to continue our screening protocol to find additional mutants that exhibit dysfunctions in these processes. In Aim 2, we propose to characterize the mechanisms by which these mutations alter axon development. In Aim 3, we propose to map and identify molecular lesions that underlie mutant phenotypes indicative of defects in axon transport and/or microtubule dynamics. Our approach is innovative, because it combines the genetic advantages of the zebrafish system with new imaging approaches to define the roles of axon transport and microtubule dynamics during neural development. Public health relevance: Development of this vertebrate system, in which we can study the mechanisms of axon transport in an intact animal, will expedite the elucidation of causative factors of neurodevelopmental and neurodegenerative disorders whose etiologies lie in disruption of axon transport and/or microtubule dynamics.

描述(申请人提供)：在神经系统发育和维持过程中，细胞内蛋白质/细胞器运输和微管动力学的协调在轴突生长和回路形成中起着至关重要的作用。这些突起的异常与许多神经发育和神经退行性疾病有关，如Charcot-Marie-Tooth病、阿尔茨海默病和肌萎缩侧索硬化症。尽管这些疾病普遍存在，但目前还没有有效的治疗方法。这种缺陷可以部分归因于体内脊椎动物系统的缺乏，而这是必需的 有效地分析轴突运输或微管动力学的改变如何导致轴突延伸失败或轴突变性。我们的长期目标是利用斑马鱼作为活体模型系统，确定调控轴突运输和微管动力学的细胞和分子机制的遗传基础。这项建议的总体目标是使用正向遗传筛查来寻找在脊椎动物神经系统发育过程中调节轴突运输和微管动力学的因素。我们的初步研究证实，这一筛选是可行的。我们进行了中试筛选，产生了5个突变株，表现出与轴突运输缺陷和/或微管动力学障碍一致的表型，包括：轴突截断、神经变性和轴突肿胀。我们还开发了一些分析方法来表征我们突变品系中微管-马达介导的运输和细胞骨架动力学，包括在完整的斑马鱼胚胎和幼体中可视化这些过程的成像方法。使用这些方法，我们已经证明了其中一个突变体(Llg1)，它包含JNK相互作用蛋白3(JIP3)的一个同源突变，在轴突运输方面存在特定的缺陷，同时显示出异常的微管动力学。在目标1中，我们建议继续我们的筛选方案，以寻找在这些过程中表现出功能障碍的其他突变体。在目标2中，我们建议描述这些突变改变轴突发育的机制。在目标3中，我们建议定位和识别突变表型下的分子损伤，这些突变表型表明轴突运输和/或微管动力学方面的缺陷。我们的方法是创新的，因为它结合了斑马鱼系统的遗传优势和新的成像方法，以确定轴突运输和微管动力学在神经发育中的作用。公共卫生相关性：开发这一脊椎动物系统，我们可以在其中研究完整动物的轴突运输机制，将加快阐明神经发育和神经退行性疾病的病因，这些疾病的原因在于轴突运输和/或微管动力学的破坏。