In vivo analysis of the mechanisms of axon transport.

轴突运输机制的体内分析。

基本信息

批准号：
8125867
负责人：
Catherine M Drerup
金额：
$ 5.31万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-05-01 至 2012-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8125867
关键词：
Accounting Address Affect Alzheimer&apos s Disease Amyotrophic Lateral Sclerosis Animals Axon Axonal Transport Binding Biological Assay Biological Models Carrier Proteins Cell Culture Techniques Cell physiology Charcot-Marie-Tooth Disease Cytoskeleton Data Defect Development Disease Distal Dynein ATPase Embryo Etiology Exhibits Genes Genetic Genetic Screening Goals Growth Health Human Image In Vitro Interruption Kinesin Knowledge Label Lesion Life Light Maintenance Mediating Microtubules Modeling Molecular Molecular Motors Motor Movement N-terminal Nature Nerve Nervous system structure Neurodegenerative Disorders Neurodevelopmental Disorder Neurons Nonsense Codon Organelles Pathology Phenotype Phosphotransferases Presynaptic Terminals Process Proteins Regulation Reporting Sensory Signal Pathway Spinal Muscular Atrophy Swelling Synapses System Techniques Testing Therapeutic Travel Vertebrates Work Zebrafish base density effective therapy fly gene function human disease in vivo in vivo Model insight mutant neural circuit neuronal cell body novel novel strategies positional cloning relating to nervous system research study synaptogenesis tau Proteins

项目摘要

DESCRIPTION (provided by applicant): In neurons, axonal transport of proteins and organelles to and from synapses is essential for formation and maintenance of neural connectivity. Impaired axon transport is thought to contribute to numerous neurodevelopmental and neurodegenerative disorders, including Alzheimer's Disease, Amyotrophic Lateral Sclerosis, and Charcot-Marie Tooth Disease. Despite the pervasiveness of these disorders, their underlying causes are still poorly understood, which has hindered the development of effective therapies This is at least partially due to the lack of a vertebrate model system in which to study this process in vivo and test potential therapeutics. I have developed zebrafish as an in vivo model for studying axon transport by 1) developing a novel imaging approach to visualize movement of fluorescently labeled cargo in an intact animal; and 2) participating in a forward genetic screen to isolate mutants with axonal transport defects. One of these mutant strains (rogue) has a phenotype typical of disruptions in axon transport, i.e. nerve truncation, nerve thinning and distal axonal swellings in long sensory axons. Live imaging revealed that rogue has reduced density and altered transport parameters of some actively transported cargos. Positional cloning identified the underlying genetic lesion in the gene encoding jnk interacting protein 3 (jip3). Previous studies in vitro revealed that Jip3 binds both the microtubule motor Kinesin-1 and axonal cargo. Jip3 has also been shown to modulate cJun N- terminal kinase (Jnk) activity in cell culture, which could potentially have downstream effects on the microtubule cytoskeleton and cargo-motor binding. However, which axonal cargos, if any, are directly Jip3- dependent and which of these Jip3-dependent molecular interactions regulate axonal transport during axon extension and synapse formation is not known. To address these questions, I will first use the live embryo imaging approach I developed to determine if microtubule dynamics and axon transport of specific cargos are disrupted in rogue. Second, I will determine if Jip3 interaction with Jnk and/or Kinesin-1 are necessary for proper regulation of the microtubule cytoskeleton or axonal transport of specific cargos, thus promoting axon extension and synapse formation. The proposed experiments will define the Jip3-dependent cellular and molecular processes which mediate axon transport in vivo. Long-term, the system I have developed can be used to analyze axon transport in vivo to fully understand how abnormalities in this process disrupt nervous system formation and function in normal and disease states. PUBLIC HEALTH RELEVANCE: The transport of proteins and organelles from the neuronal cell body to axon terminals and vice versa is critical both to maintain the health of the cell body and support the formation of functional nervous system connections. Defects in this process are associated with numerous developmental and neurodegenerative diseases such as Spinal Muscular Atrophy, Alzheimer's Disease, and Amyotrophic Lateral Sclerosis. The knowledge gained from these studies will advance our understanding of the basic mechanisms required for axonal transport in vivo. Additionally, they will establish zebrafish as a model system that can be used to investigate the function of genes associated with axonal diseases to determine if disease etiology lies in interruptions of this basic cellular process.

描述（由申请人提供）：在神经元中，蛋白质和细胞器进出突触的轴突运输对于神经连接的形成和维持至关重要。轴突运输受损被认为会导致许多神经发育和神经退行性疾病，包括阿尔茨海默病、肌萎缩侧索硬化症和腓骨肌萎缩症。尽管这些疾病普遍存在，但对其根本原因仍知之甚少，这阻碍了有效疗法的开发。这至少部分是由于缺乏脊椎动物模型系统来研究体内这一过程并测试潜在的疗法。我开发了斑马鱼作为研究轴突运输的体内模型，方法是：1）开发一种新颖的成像方法来可视化完整动物中荧光标记货物的运动； 2) 参与正向遗传筛选以分离具有轴突运输缺陷的突变体。其中一种突变株（流氓）具有轴突运输中断的典型表型，即神经截断、神经变薄和长感觉轴突的远端轴突肿胀。实时成像显示，流氓降低了一些主动运输货物的密度并改变了运输参数。定位克隆鉴定了编码 jnk 相互作用蛋白 3 (jip3) 的基因中潜在的遗传病变。先前的体外研究表明，Jip3 结合微管运动驱动蛋白-1 和轴突货物。 Jip3 还被证明可以调节细胞培养中的 cJun N 末端激酶 (Jnk) 活性，这可能对微管细胞骨架和货物马达结合产生下游影响。然而，哪些轴突货物（如果有的话）直接依赖于 Jip3，以及哪些依赖于 Jip3 的分子相互作用在轴突延伸和突触形成过程中调节轴突运输尚不清楚。为了解决这些问题，我将首先使用我开发的活胚胎成像方法来确定流氓中特定货物的微管动力学和轴突运输是否被破坏。其次，我将确定 Jip3 与 Jnk 和/或 Kinesin-1 的相互作用对于正确调节微管细胞骨架或特定货物的轴突运输是否是必要的，从而促进轴突延伸和突触形成。拟议的实验将定义介导体内轴突运输的 Jip3 依赖性细胞和分子过程。从长远来看，我开发的系统可用于分析体内轴突运输，以充分了解该过程中的异常如何扰乱正常和疾病状态下的神经系统形成和功能。公共健康相关性：蛋白质和细胞器从神经元细胞体到轴突末端的运输（反之亦然）对于维持细胞体的健康和支持功能性神经系统连接的形成至关重要。这一过程的缺陷与许多发育和神经退行性疾病有关，例如脊髓性肌萎缩症、阿尔茨海默病和肌萎缩侧索硬化症。从这些研究中获得的知识将增进我们对体内轴突运输所需基本机制的理解。此外，他们还将建立斑马鱼作为模型系统，可用于研究与轴突疾病相关的基因功能，以确定疾病病因是否在于这一基本细胞过程的中断。