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

项目总结/摘要 肌萎缩侧索硬化症（ALS）是一种涉及运动神经元变性的致死性疾病。死亡发生3-5 诊断后数年，没有治愈的方法，而有限的治疗方法只能延长生存时间， 个月ALS发病机制对研究人员来说仍然是难以捉摸的;科学家们仍然不确定 到底是什么导致运动神经元中毒死亡在本建议书中，我们将调查 肌动蛋白动力学调节缺陷在ALS中发挥的机制作用。 这一研究过程受到Profilin 1（PFN 1）突变的发现的刺激，Profilin 1（PFN 1）是一种关键的调节因子， 细胞骨架动力学，抑制其结合肌动蛋白的能力，负责约1-2%的家族性肌萎缩性 侧索硬化症（fALS）。过表达这些突变PFN 1构建体的运动神经元表现出轴突生长抑制， 生长和异常肌动蛋白细胞骨架。PFN 1突变作为致病因子的鉴定 fALS提出了一个令人兴奋的新假设，即肌动蛋白细胞骨架动力学在细胞内 维持运动神经元的健康和肌动蛋白动力学的损伤，随着时间的推移， 神经变性由于Pfn 1是一种G-肌动蛋白结合蛋白，空间定位G-肌动蛋白的过程， 调节丝聚合和G-/F-肌动蛋白（G/F）比率是特别感兴趣的。 我们假设PFN 1下游的肌动蛋白细胞骨架动力学缺陷，如G-肌动蛋白定位， 在ALS发病机制中起着至关重要的作用。为了确定受损的肌动蛋白动力学是否是ALS的标志， 为探讨Pfn 1突变诱导fALS的机制，我们提出以下具体目的：（1） 确定G-actin动态调节中的缺陷在ALS中所起的作用;（2）确定特定的 ALS连锁的PFN 1突变体如何改变肌动蛋白动力学的机制;和（3）确定特定的细胞 G-actin动态定位机制及其在运动神经元生长和发育中的作用 上维护 我们将研究肌动蛋白动力学使用高分辨率定量成像在运动神经元和神经- 肌萎缩侧索硬化症小鼠模型的肌肉组织我们还将研究肌动蛋白在功能性运动神经元衍生 从人类ALS患者的诱导多能干细胞中提取。最近，我们发现了一种新的途径， 肌动蛋白的时空定位，以调节细胞的运动和轴突的指导。因此，我们设计了一个 许多独特的测定，以可视化G-肌动蛋白定位，计算G/F肌动蛋白比，并定量肌动蛋白 迁移率本建议中所提出的问题将使人们更深入地了解 动力学在运动神经元发育和维持突触前末梢中的作用 神经肌肉接头，以及确定肌动蛋白调节缺陷可能导致ALS的方式。

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.