Selectivity for Kinesin-driven Transport of Axonal RNA Granules

驱动蛋白驱动的轴突 RNA 颗粒运输的选择性

• 批准号: 9791030
• 负责人: Yusuke Fukuda
• 金额: $ 10.66万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-30 至 2020-05-22
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9791030
• 关键词: Afferent Neurons; Alternative Splicing; Amyotrophic Lateral Sclerosis; Area; Axon; Axonal Transport; Binding; Bypass; Cells; Characteristics; Charcot-Marie-Tooth Disease; Complex; Consensus; Cytoplasmic Granules; Data; Defect; Degenerative Disorder; Dendrites; Development Plans; Disease; Disease model; Distal; Dynein ATPase; Environment; Family; Genes; Glutamine; Goals; Grant; Hereditary Motor and Sensory-Neuropathy Type II; Hereditary Spastic Paraplegia; Imaging Techniques; Impairment; In Vitro; Individual; Interruption; Intracellular Transport; Investigation; Kinesin; Knockout Mice; Lead; Life; Light; Mass Spectrum Analysis; Mediating; Microtubules; Modeling; Molecular; Motor; Motor Neurons; Movement; Mutate; Mutation; Neurodegenerative Disorders; Neurons; Neurosciences; Paclitaxel; Pathway interactions; Peptides; Peripheral Nervous System Diseases; Post-Translational Protein Processing; Proline; Protein Isoforms; Proteins; Proteome; Proteomics; Public Health; RNA; RNA Splicing; RNA Transport; RNA-Binding Proteins; Regulation; Research; Role; Specific qualifier value; Specificity; Spinal Ganglia; System; Tail; Testing; Therapeutic; Therapeutic Intervention; Training; Transcript; Translating; Translations; Transport Process; Zebrafish; anterograde transport; axonal degeneration; base; career development; design; experimental study; human disease; in vitro testing; in vivo; in vivo Model; innovation; insight; live cell imaging; medical schools; member; mimetics; multidisciplinary; mutant; nervous system disorder; neuronal cell body; novel; novel therapeutic intervention; novel therapeutics; peptidomimetics; prevent; protein expression; therapeutic evaluation; transcriptome sequencing; transcriptomics

Intracellular transport is critical for the function and viability of neurons throughout life. Splicing factor proline- glutamine rich (SFPQ) is an RNA-binding protein that packages trophic-regulated transcripts, such as Bclw, into RNA granules (RNAGs). Local translation of these transcripts is critical for protecting axons from degeneration. The objective of this grant is to understand the mechanism by which SFPQ RNAGs are localized to axons and translated there. This investigation builds on my key findings that: 1) SFPQ specifically binds to only one of three members of the kinesin-1 family of motors, KIF5A, and to only one of the associated kinesin light chains (KLC), KLC1; 2) a newly defined consensus EDxYxE motif within the coiled coil (CC) region of SFPQ is required for binding to KIF5A/KLC1; and 3) the variable carboxy-terminal tail (CTT) region of KIF5A is required for binding to SFPQ. These data demonstrating selectivity of the kinesins is highly relevant to human disease as KIF5A is the only kinesin-1 motor that is mutated in Charcot-Marie-Tooth disease (CMT), hereditary spastic paraplegia (HSP) and in amyotrophic lateral sclerosis (ALS). Moreover, ALS mutations in SFPQ lie within the CC region adjacent to EDxYxE motif. Together, I propose a CENTRAL HYPOTHESIS that SFPQ RNAGs are localized to axons through a highly specific KIF5A/KLC1-dependent transport and that disruption of this pathway results in KIF5A and SFPQ-related neurological diseases. In this proposal I will test the following predictions of this Hypothesis: 1) anterograde transport of SFPQ depends on interactions mediated by the CTT of KIF5A and by KLC1; 2) axonal survival requires KIF5A-mediated transport of SFPQ RNAGs to axons; and 3) Bclw mimetics can prevent axonal degeneration caused by interruption of KIF5A-mediated transport of SFPQ. These 3 Aims will reveal mechanistic understanding of how defect in specific kinesin-driven transport characteristically leads to axon degeneration in neurological disease and will assess the therapeutic potential of a highly innovative Bclw peptide. I have designed an effective training plan to execute this proposal and to advance in 4 specialized training areas: 1) compartmented neuronal culture system to study spatial regulation of protein expression; 2) advanced quantitative live cell imaging techniques in axons; 3) transcriptomics and proteomics to profile and determine regulatory mechanism of specialized motor adaptor complex formation by alternative splicing and post-translational modifications; and 4) use of in vivo disease models for therapeutic intervention. My career development plan is designed to be highly collaborative; several advisors are readily available within the multi-disciplinary environment of the greater Harvard Medical School campus. Upon conclusion I will initiate the first step towards my overarching goal in understanding how defects in microtubule- based transport in neurons lead to neurological diseases; why mutations in a specific motor component cause degeneration in neurons; and to bridge basic neuroscience discovery into new therapeutics against neurological diseases of sensory and motor neurons including CMT, HSP and ALS.

细胞内转运对神经元的功能和生存能力至关重要。剪接因子脯氨酸- 富含谷氨酰胺（SFPQ）是一种RNA结合蛋白，其包装营养调节转录物，如Bclw， RNA颗粒（RNAGs）。这些转录本的局部翻译对于保护轴突免受 退化这项资助的目的是为了了解SFPQ RNAG的本地化机制 到轴突并在那里翻译。这项研究建立在我的主要发现之上：1）SFPQ特异性结合 驱动蛋白-1家族的三个成员之一，KIF 5A，和相关的驱动蛋白之一， 轻链（KLC），KLC 1; 2）在卷曲螺旋（CC）区域内新定义的共有EDxYxE基序， SFPQ是与KIF 5A/KLC 1结合所必需的;和3）KIF 5A的可变羧基末端尾（CTT）区是 需要绑定到SFPQ。这些数据表明驱动蛋白的选择性与人类高度相关。 KIF 5A是唯一的在Charcot-Marie-Tooth病（CMT）中突变的驱动蛋白-1马达，遗传性 痉挛性截瘫（HSP）和肌萎缩侧索硬化症（ALS）。此外，SFPQ中的ALS突变与 在与EDxYxE基序相邻的CC区内。总之，我提出了一个中心假设，即SFPQ RNAG通过高度特异性的KIF 5A/KLC 1依赖性转运定位于轴突， 该途径导致KIF 5A和SFPQ相关的神经疾病。在本提案中，我将测试以下内容 该假说的预测：1）SFPQ的顺行转运依赖于CTT介导的相互作用 2）轴突存活需要KIF 5A介导的SFPQ RNAG向轴突的转运;以及 3)Bclw模拟物可以预防由KIF 5A介导的Bclw转运中断引起的轴突变性。 SFPQ。这3个目的将揭示在特定驱动蛋白驱动的运输中缺陷是如何机制性地理解的。 特征性地导致神经系统疾病中的轴突变性， 一种高度创新的Bclw肽我已经设计了一个有效的培训计划来执行这个建议， 在4个专业培训领域取得进展：1）研究空间调节的隔室神经元培养系统 2）先进的轴突定量活细胞成像技术; 3）转录组学和 蛋白质组学分析和确定专门的马达适配器复合物形成的调节机制， 选择性剪接和翻译后修饰;和4）体内疾病模型用于治疗的用途 干预我的职业发展计划旨在高度协作;几位顾问随时准备 在大哈佛医学院校园的多学科环境中提供。后 结论我将开始我的首要目标的第一步，在了解如何在微管缺陷- 神经元的基础运输导致神经系统疾病;为什么特定运动成分的突变会导致 神经元变性;并将基础神经科学发现与新的治疗方法联系起来， 感觉和运动神经元的神经系统疾病，包括CMT、HSP和ALS。