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.

细胞内转运对于一生中神经元的功能和活力至关重要。剪接因子脯氨酸 - 富含谷氨酰胺（SFPQ）是一种RNA结合蛋白，包装营养调节的转录本，例如BCLW， 进入RNA颗粒（RNAGS）。这些成绩单的局部翻译对于保护轴突免受 退化。这笔赠款的目的是了解SFPQ rnag局部的机制 到轴突并在那里翻译。这项调查以我的关键发现为基础：1）SFPQ专门绑定到 动机1电动机家族KIF5A的三个成员中只有一个，只有一个相关的驱动蛋白 轻链（KLC），KLC1； 2）在盘绕线圈（CC）区域内的新定义的共识EDXYXE图案 与KIF5A/KLC1结合需要SFPQ； 3）KIF5A的可变羧基末端尾部（CTT）区域为 与SFPQ结合所需。这些证明驱动素选择性的数据与人类高度相关 作为KIF5A的疾病是唯一在Charcot-Marie-Tooth疾病（CMT）中突变的动力蛋白-1电动机 痉挛性截瘫（HSP）和肌萎缩性侧索硬化症（ALS）。而且，SFPQ中的ALS突变谎言 在与Edxyxe基序相邻的CC区域内。我一起提出了一个中心假设，即SFPQ RNAG通过高度特异 该途径导致KIF5A和与SFPQ相关的神经系统疾病。在此提案中，我将测试以下内容 该假设的预测：1）SFPQ的顺行转运取决于CTT介导的相互作用 kif5a和klc1; 2）轴突存活需要KIF5A介导的SFPQ rnags向轴突的转运；和 3）BCLW Mimetics可以防止由KIF5A介导的转运中断引起的轴突变性 SFPQ。这三个目标将揭示对特定运动蛋白驱动运输中缺陷的机理理解 典型地导致神经疾病的轴突变性，并将评估治疗潜力 高度创新的BCLW肽。我设计了一个有效的培训计划来执行此建议，并 在4个专业培训领域的进步：1）研究空间调节的隔室神经元培养系统 蛋白质表达； 2）轴突中的高级定量活细胞成像技术； 3）转录组学和 蛋白质组学以剖析并确定专业运动适配器复合物形成的调节机制 替代剪接和翻译后修饰； 4）使用体内疾病模型进行治疗 干涉。我的职业发展计划旨在高度协作；几位顾问很容易 在大哈佛医学院校园的多学科环境中可用。之上 结论我将迈出朝着我的总体目标迈出的第一步，以了解微管中的缺陷如何 基于神经元的运输导致神经疾病；为什么特定电机组件中的突变原因 神经元的变性；并将基本神经科学发现桥接成新的治疗剂 感官和运动神经元的神经疾病，包括CMT，HSP和ALS。