In vivo analysis of the mechanisms of axon transport.

描述（由申请人提供）：在神经元中，蛋白质和细胞器在突触之间的轴突运输对于神经连通性的形成和维持至关重要。轴突运输受损被认为是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、肌萎缩性侧索硬化症和沙克-玛丽牙病。尽管这些疾病普遍存在，但其潜在原因仍然知之甚少，这阻碍了有效治疗方法的发展，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治疗方法。我已经开发了斑马鱼作为体内模型来研究轴突运输：1)开发了一种新的成像方法来可视化荧光标记货物在完整动物中的运动；2)参与前向基因筛选，分离轴突转运缺陷突变体。其中一种突变株（流氓）具有典型的轴突运输中断表型，即神经截断，神经变薄和长感觉轴突远端轴突肿胀。实时成像显示流氓降低了一些正在运输的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）的基因的潜在遗传损伤。先前的体外研究表明，Jip3可以结合微管运动蛋白激酶-1和轴突货物。jp3也被证明在细胞培养中调节cJun N-末端激酶（Jnk）活性，这可能对微管细胞骨架和货物-马达结合有潜在的下游影响。然而，哪些轴突货物（如果有的话）是直接依赖于Jip3的，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微管动力学和特定货物的轴突运输是否在流氓中被破坏。其次，我将确定Jip3与Jnk和/或Kinesin-1的相互作用对于适当调节微管细胞骨架或特定货物的轴突运输是否必要，从而促进轴突延伸和突触形成。提出的实验将确定介导轴突运输的jip3依赖的细胞和分子过程。从长远来看，我开发的系统可以用来分析体内轴突运输，以充分了解这一过程中的异常如何在正常和疾病状态下破坏神经系统的形成和功能。

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.