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

项目摘要/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命疾病。死亡发生3-5次 确诊数年后，没有治愈的方法，有限的治疗方法只会延长生存时间 月份。肌萎缩侧索硬化症的发病机制对研究人员来说仍然难以捉摸；科学家仍然不确定 究竟是什么导致运动神经元中毒并死亡。在这项建议中，我们将调查 肌动蛋白动力学调节缺陷在肌萎缩侧索硬化症中的作用。 这一调查过程是由发现Profilin1(PFN1)的突变所推动的，Profilin1(PFN1)是 细胞骨架动力学，抑制其结合肌动蛋白的能力，是大约1-2%的家族性肌营养不良的原因 侧索硬化症(FALS)。过度表达这些突变型PFN1结构的运动神经元显示出抑制轴突 生长并有异常的肌动蛋白细胞骨架。新城疫病原菌PFN1基因突变的鉴定 FALS提出了一个令人兴奋的新假说，即肌动蛋白细胞骨架动力学在 随着时间的推移，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 神经退行性变。由于PFN1是一种G-肌动蛋白结合蛋白，因此将G-肌动蛋白空间定位到 调节细丝聚合和G-/F-肌动蛋白(G/F)比率是特别有意义的。 我们假设PFN1下游的肌动蛋白细胞骨架动力学缺陷，如G-肌动蛋白定位， 在肌萎缩侧索硬化症的发病机制中起关键作用。以确定肌动蛋白动力学受损是否是ALS的标志，并 为了探讨PFN1基因突变导致FALS的机制，我们提出了以下具体目标：(1) 确定缺陷在ALS中G-肌动蛋白动态调节中的作用；(2)确定特异性 ALS连锁的PFN1突变体如何改变肌动蛋白动力学的机制；以及(3)确定特定的细胞 G-肌动蛋白动态定位机制及其在运动神经元生长调控中的作用 维修。 我们将使用高分辨率定量成像来研究运动神经元和神经中的肌动蛋白动力学- 肌萎缩侧索硬化症小鼠模型的肌肉外植体。我们还将研究功能性运动神经元中的肌动蛋白。 来自人类肌萎缩侧索硬化症患者的诱导多能干细胞。最近，我们发现了一种新的途径，其中 G-肌动蛋白在时空上定位于调控细胞运动和轴突引导。因此，我们设计了一个 显示G-肌动蛋白定位、计算G/F肌动蛋白比率和量化肌动蛋白的独特分析方法的数量 机动性。本提案中提出的问题将使我们对肌动蛋白的作用有更深的理解 运动神经元发育和突触前终末维持中的动力学作用 神经肌肉连接，以及确定肌动蛋白调节缺陷可能导致肌萎缩侧索硬化症的途径。

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.