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（由申请人提供）：在神经系统发育和维持期间，细胞内蛋白质/细胞器转运和微管动力学的协调在轴突生长和回路形成中起重要作用。这些过程中的异常与许多神经发育和神经退行性疾病相关，例如Charcot-Marie-Tooth病、阿尔茨海默病和肌萎缩性侧索硬化症。尽管这些疾病普遍存在，但没有有效的治疗方法。这种缺陷可以部分归因于缺乏体内脊椎动物系统，这是需要的。 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。我们的长期目标是确定的细胞和分子机制，调节轴突运输和微管动力学使用斑马鱼作为体内模型系统的遗传基础。这项建议的总体目标是使用正向遗传筛选，以找到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，这种筛选是可行的。我们进行了一个试点筛选，产生5突变株表现出的表型与有缺陷的轴突运输和/或破坏微管动力学，包括：轴突截断，神经变性和轴突software一致。我们还开发了一些检测方法来表征我们突变株中微管马达介导的运输和细胞骨架动力学，包括成像方法来可视化完整斑马鱼胚胎和幼虫中的这些过程。使用这些方法，我们已经证明，其中一个突变体（llg 1），其中包含一个突变的JNK相互作用蛋白3（Jip 3）的直系同源物具有特定的缺陷，轴突运输，同时表现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，以找到在这些过程中表现出功能障碍的其他突变体。在目标2中，我们提出了这些突变改变轴突发育的机制。在目标3中，我们提出映射和识别的突变体表型指示轴突运输和/或微管动力学的缺陷的基础上的分子病变。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与新的成像方法来定义轴突运输和微管动力学在神经发育过程中的作用。公共卫生相关性：这种脊椎动物系统的发展，我们可以在一个完整的动物轴突运输的机制进行研究，将加快阐明神经发育和神经退行性疾病的病因在于轴突运输和/或微管动力学的破坏因素。