In vivo analysis of the mechanisms of axon transport.

轴突运输机制的体内分析。

• 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
• 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' 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

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(申请人提供)：在神经元中，蛋白质和细胞器的轴突运输进出突触对于神经连接的形成和维持是必不可少的。轴突运输受损被认为是导致许多神经发育和神经退行性疾病的原因，包括阿尔茨海默病、肌萎缩侧索硬化症和夏科-玛丽牙病。尽管这些疾病普遍存在，但其根本原因仍然鲜为人知，这阻碍了有效疗法的开发这至少部分是由于缺乏在体内研究这一过程并测试潜在疗法的脊椎动物模型系统。我开发了斑马鱼作为研究轴突运输的活体模型，方法是：1)开发一种新的成像方法，以可视化完整动物中荧光标记的货物的运动；2)参与正向遗传筛查，以分离具有轴突运输缺陷的突变体。其中一个突变株(ROGGE)具有典型的轴突运输中断的表型，即神经截断、神经变薄和长感觉神经轴突的远端轴突肿胀。现场成像显示，流氓降低了一些活跃运输货物的密度，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)基因的潜在遗传损伤。先前的体外研究表明，JIP3既能结合微管运动蛋白-1，又能结合轴突货物。JIP3还被证明可以调节细胞培养中cJun氨基末端激酶(JNK)的活性，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，在轴突延伸和突触形成过程中，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特定货物的微管动力学和轴突运输是否在无赖状态下被破坏。其次，我将确定JIP3与JNK和/或Kinesin-1的相互作用是否是适当调节微管细胞骨架或特定货物的轴突运输，从而促进轴突延伸和突触形成所必需的。拟议中的实验将定义依赖JIP3的细胞和分子过程，这些过程在体内介导轴突运输。从长远来看，我开发的系统可以用来分析体内轴突的运输，以充分了解这一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。 公共卫生相关性：蛋白质和细胞器从神经元细胞体到轴突终末的运输，反之亦然，对于维持细胞体的健康和支持神经系统功能连接的形成都是至关重要的。这一过程中的缺陷与许多发育和神经退行性疾病有关，如脊髓肌肉萎缩、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促进我们对体内轴突运输所需的基本机制的理解。此外，他们将建立斑马鱼作为一个模型系统，可以用来研究与轴突疾病相关的基因的功能，以确定疾病的病因是否在于这一基本细胞过程的中断。