Novel mechanisms of actin dynamics underlying cell motility, axon growth, and ALS

细胞运动、轴突生长和 ALS 背后肌动蛋白动力学的新机制

• 批准号: 9042507
• 负责人: Eric A Vitriol
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-15 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9042507
• 关键词: Actin-Binding Protein; Actins; Address; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Axon; Binding; Binding Proteins; Biological Assay; Biology; Brain; Cells; Cessation of life; Cognition; Computer Analysis; Cytoskeleton; Defect; Development; Diagnosis; Diffuse; Disease; F-Actin; Familial Amyotrophic Lateral Sclerosis; Filament; G Actin; Growth; Growth Cones; Health; Human; Huntington Disease; Image; Impairment; Induced Mutation; Investigation; Lead; Life; Link; Maintenance; Membrane; Microfilaments; Microscopy; Motor; Motor Neurons; Muscle; Mutation; Nerve; Nerve Degeneration; Nervous system structure; Neurodegenerative Disorders; Neuromuscular Junction; Neurons; Organism; Pathogenesis; Pathway interactions; Patients; Phase; Play; Presynaptic Terminals; Process; Regulation; Regulatory Pathway; Relative (related person); Research; Research Personnel; Resolution; Role; Scientist; Source; Spinal Ganglia; Testing; Time; Training; abstracting; axon growth; axon guidance; base; cell motility; cellular imaging; design; induced pluripotent stem cell; interest; monomer; motor neuron degeneration; motor neuron development; mouse model; mutant; neurodevelopment; novel; novel diagnostics; overexpression; polymerization; presynaptic neurons; programs; quantitative imaging; spatiotemporal; tool

Project Summary/Abstract Amyotrophic lateral sclerosis (ALS) is a fatal disease involving motor neuron degeneration. Death occurs 3-5 years after diagnosis, there is no cure, and what limited treatments do exist only extend survival by a matter of months. The mechanism of ALS pathogenesis has remained elusive to researchers; scientists are still unsure exactly what causes the motor neurons to become toxic and die. In this proposal, we will investigate the mechanistic role that defects in the regulation of actin dynamics plays in ALS. This course of investigation was spurred by the discovery that mutations in Profilin1 (PFN1), a key regulator of cytoskeletal dynamics, that inhibit its ability to bind actin are responsible for about 1-2% of familial Amyotrophic lateral sclerosis (fALS). Motor neurons overexpressing these mutant PFN1 constructs displayed inhibited axon growth and had abnormal actin cytoskeletons. The identification of PFN1 mutations as causative agents in fALS presents an exciting new hypothesis that actin cytoskeletal dynamics play a fundamental role in maintaining the health of motor neurons and that impairment of actin dynamics could, over time, lead to neurodegeneration. Because Pfn1 is a G-actin binding protein, the processes that spatially localize G-actin to regulate filament polymerization and the G-/F-actin (G/F) ratio are of particular interest. We hypothesize that defects in actin cytoskeletal dynamics downstream of PFN1, such as G-actin localization, play a crucial role in ALS pathogenesis. To determine if impaired actin dynamics are a hallmark of ALS and to investigate the mechanism of how Pfn1 mutations induce fALS, we propose the following Specific Aims: (1) Determine the role that defects in the dynamic regulation of G-actin plays in ALS; (2) Determine the specific mechanism of how ALS-linked PFN1 mutants alter actin dynamics; and (3) Determine the specific cellular mechanism of dynamic G-actin localization and its function in regulating motor neuron growth and maintenance. We will investigate actin dynamics using high-resolution quantitative imaging in motor neurons and nerve- muscle explants from mouse models of ALS. We will also examine actin in functional motor neurons derived from induced pluripotent stem cells from human ALS patients. Recently, we discovered a novel pathway where G-actin was spatiotemporally localized to regulate cell motility and axon guidance. Thus, we have designed a number of unique assays to visualize G-actin localization, calculate the G/F actin ratio, and quantify actin mobility. The questions addressed in this proposal will yield a deeper understanding of the role that actin dynamics play in motor neuron development and maintenance of the presynaptic terminal of the neuromuscular junction, as well as identify ways that defects in actin regulation can cause ALS.

项目摘要/摘要 肌萎缩性外侧硬化症（ALS）是一种致命疾病，涉及运动神经元变性。死亡发生3-5 诊断后的几年，无法治愈，并且实际上存在有限的治疗方法，只有将生存扩展到 月份。 ALS发病机理的机制仍然难以捉摸。科学家仍然不确定 正是导致运动神经元变得有毒并死亡的原因。在此提案中，我们将调查 在ALS中肌动蛋白动力学调节中缺陷的机械作用。 发现profilin1中的突变（PFN1）刺激了这一调查过程， 抑制其结合肌动蛋白的能力的细胞骨架动力学负责大约1-2％的家族性肌萎缩症 侧硬化症（fals）。过表达这些突变体PFN1构建体的运动神经元显示出抑制的轴突 生长并具有异常的肌动蛋白细胞骨架。将PFN1突变鉴定为病因 伪造提出了令人兴奋的新假设，即肌动蛋白细胞骨架动力学在 维持运动神经元的健康和肌动蛋白动力学的损害可能会导致 神经变性。因为PFN1是一种G-肌动蛋白结合蛋白，因此在空间上将G-肌动蛋白定位到 调节细丝聚合和g/f-肌动蛋白（g/f）的比率特别感兴趣。 我们假设PFN1下游肌动蛋白细胞骨架动力学的缺陷，例如G-肌动蛋白定位， 在ALS发病机理中起关键作用。确定受损肌动蛋白动力学是否是ALS的标志 研究PFN1突变如何诱导伪造的机制，我们提出以下特定目的：（1） 确定缺陷在ALS中G-肌动蛋白发挥的动态调节中的作用； （2）确定特定 ALS连接的PFN1突变体如何改变肌动蛋白动力学的机制； （3）确定特定的细胞 动态G-肌动蛋白定位的机理及其在调节运动神经元生长和 维护。 我们将使用运动神经元中的高分辨率定量成像研究肌动蛋白动力学 来自ALS小鼠模型的肌肉外植体。我们还将检查衍生的功能运动神经元中的肌动蛋白 来自人类ALS患者的诱导多能干细胞。最近，我们发现了一条新颖的道路 G-肌动蛋白的时空定位以调节细胞运动和轴突引导。因此，我们设计了 可视化G-肌动蛋白定位，计算G/F肌动蛋白比并量化肌动蛋白的独特测定数量 机动性。本提案中解决的问题将对肌动蛋白的作用有更深入的了解 运动神经元开发和维持突触前终端的动态发挥 神经肌肉结，以及确定肌动蛋白调节缺陷的方法可能引起ALS。