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（由申请人提供）：在神经元中，蛋白质和细胞器与突触的轴突运输对于形成和维持神经连通性至关重要。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，包括阿尔茨海默氏病，肌萎缩性侧面硬化症和charcot-marie牙齿疾病。尽管这些疾病普遍存在，但它们的根本原因仍然很少了解，这阻碍了有效疗法的发展，这至少部分是由于缺乏脊椎动物模型系统在体内研究这一过程并测试潜在的治疗剂。我已经开发了斑马鱼作为一个体内模型，用于研究轴突传输1）开发一种新型的成像方法来可视化完整动物中荧光标记的货物的运动； 2）参与前遗传筛选，以分离出具有轴突转运缺陷的突变体。这些突变菌株之一（流氓）具有轴突转运中典型的干扰表型，即长感觉轴突中的神经截断，神经变薄和远端轴突肿胀。实时成像表明，流氓已经降低了某些积极运输的嘉戈斯的密度和转运参数的改变。位置克隆确定了编码JNK相互作用蛋白3（JIP3）的基因中的潜在遗传病变。先前的体外研究表明，JIP3结合了微管运动动力蛋白-1和轴突货物。 JIP3还显示出在细胞培养中调节CJUN N末端激酶（JNK）活性，这可能会对微管细胞骨架和货物运动结合产生下游影响。但是，哪种轴突符（如果有的话）是直接的JIP3依赖性，而这些JIP3依赖性分子相互作用中的哪个调节轴突延伸过程中的轴突转运和突触形成。为了解决这些问题，我将首先使用我开发的实时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流氓中破坏。其次，我将确定JIP3与JNK和/或驱动蛋白-1的相互作用对于适当调节特定嘉戈斯的微管细胞骨架或轴突转运是必要的，从而促进轴突延伸和突触形成。提出的实验将定义JIP3依赖性细胞和分子过程，这些过程介导体内轴突转运。长期，我开发的系统可用于分析体内的轴突运输，以充分了解此过程中的异常如何破坏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。此过程中的缺陷与许多发育和神经退行性疾病有关，例如脊柱肌肉萎缩，阿尔茨海默氏病和肌萎缩性侧向硬化症。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模型系统，可用于研究与轴突疾病相关的基因的功能，以确定疾病病因是否在于这种基本细胞过程的中断。