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

基本信息

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
项目类别：
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

项目摘要

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.

细胞内运输对于神经元一生的功能和活力至关重要。脯氨酸剪接因子- 富含谷氨酰胺 (SFPQ) 是一种 RNA 结合蛋白，可包装营养调节转录物，例如 Bclw、成 RNA 颗粒 (RNAG)。这些转录本的本地翻译对于保护轴突免受退化。这笔赠款的目的是了解 SFPQ RNAG 本地化的机制到轴突并在那里翻译。这项调查建立在我的主要发现的基础上：1) SFPQ 专门结合到仅驱动蛋白-1 马达家族的三个成员 KIF5A 之一，以及仅相关驱动蛋白之一轻链（KLC），KLC1； 2) 卷曲线圈 (CC) 区域内新定义的共有 EDxYxE 基序与 KIF5A/KLC1 结合需要 SFPQ； 3) KIF5A 的可变羧基末端尾部 (CTT) 区是绑定到 SFPQ 所需的。这些数据表明驱动蛋白的选择性与人类高度相关因为 KIF5A 是唯一在夏科-马里-图思病 (CMT) 中发生突变的驱动蛋白-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 突变位于在与 EDxYxE 基序相邻的 CC 区域内。我共同提出一个中心假设：SFPQ RNAG 通过高度特异性的 KIF5A/KLC1 依赖性运输定位于轴突，并且破坏该通路导致 KIF5A 和 SFPQ 相关的神经系统疾病。在这个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 RNAG 向轴突的转运；和 3) Bclw模拟物可以预防因KIF5A介导的细胞转运中断而引起的轴突变性 SFPQ。这三个目标将揭示对特定驱动蛋白驱动的运输中的缺陷如何发生的机制理解典型地导致神经系统疾病的轴突变性，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该建议并在 4 个专业培训领域取得进展：1）用于研究空间调节的分区神经元培养系统蛋白质表达； 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通过以下方法来分析和确定特殊运动适配器复合物形成的调节机制选择性剪接和翻译后修饰； 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；几位顾问很乐意在哈佛医学院大校园的多学科环境中可用。之上结论我将迈出实现我的总体目标的第一步，即了解微管中的缺陷如何- 神经元的基础运输导致神经系统疾病；为什么特定运动部件的突变会导致神经元退化；并将基础神经科学发现与新疗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。