Defining the mechanisms and consequences of nuclear defects in ALS/FTD

项目总结 对导致ALS/FTD神经元死亡的分子缺陷的详细洞察仍然缺乏，阻碍了 一种治疗方法的发展。核孔复合体(NPC)缺陷与核质转运 已被认为在ALS/FTD的病理过程中起核心作用。一个突出的知识差距是什么 受损的NCT对神经细胞弹性的机制、后果和致病相关性 神经退行性疾病和衰老期间。这个应用程序的目标是在机械学上识别 使新的细胞和分子通路功能失调并导致NCT失败 最终导致神经退化。特别是，我们将测试肌动蛋白细胞骨架是一种 核稳定性和NPC功能的主要调节物，ALS/FTD中这一途径的变化导致 神经元应对压力和生理刺激的能力减弱。 这项建议的具体目的是：1)定义肌动蛋白如何调节细胞的功能和功能障碍 全国人大。我们将对肌动蛋白和肌动蛋白之间的分子相互作用进行系统和深入的研究 在携带C9ORF72的IPSC来源神经元中使用药理学和遗传学方法的鼻咽癌 突变。2)确定NCT缺陷对细胞核和细胞质蛋白质组的影响 对细胞刺激的反应。通过将“组学”方法与候选方法(即CREB)相结合 途径)，我们将分析IPSC来源的神经元模型中细胞核和细胞质蛋白质组的变化 刺激后ALS/FTD的变化，我们将测试细胞骨架调制的潜力来拯救这样的 缺陷。3)确定核进口中的缺陷如何影响对细胞的转录反应 刺激。我们将分析ALS/FTD神经元基因表达和剪接图谱的变化 RNA-Seq.通过整合来自转录和转录的结果来执行网络分析 蛋白质组学方法将确定ALS/FTD相关的改变通路。细胞骨架的调制将是 用于修复已识别的缺陷。 在这项研究完成时，我们将确定控制的分子和细胞机制 全国人大的稳定和职能，以及国家技术中心的中断对全国人大的职能后果 ALS/FTD中神经元的弹性。深入了解全国人大上下游的哪些路径 在ALS/FTD中受到影响将极大地扩展我们对疾病发病机制的理解，并将使我们能够 确定尚未探索的治疗这些疾病的途径。最终，我们预计我们的研究将引领 更有效的治疗策略，利用鼻咽癌，RNA调节， 和细胞骨架。这项研究将对一系列疾病产生广泛影响，包括但不限于 肌萎缩侧索硬化症/FTD。

PROJECT SUMMARY A detailed insight into the molecular defects leading to neuronal death in ALS/FTD is still missing, hindering the development of a cure. Defects in the nuclear pore complex (NPC) and nucleocytoplasmic transport (NCT) have been proposed to play a central role in ALS/FTD pathology. An outstanding knowledge gap is what are the mechanisms, consequences, and pathogenic relevance of impaired NCT on neuronal resilience in neurodegenerative diseases and during aging. The objective of this application is to identify at the mechanistic level the novel cellular and molecular pathways that are dysfunctional and cause the failure of the NCT ultimately leading to neurodegeneration. In particular, we will test the hypothesis that the actin cytoskeleton is a main modulator of nuclear stability and NPC function, and that changes to this pathway in ALS/FTD lead to a diminished ability of neurons to cope with stress and physiological stimulation. The specific aims of this proposal are to: 1) Define how actin regulates the function and dysfunction of the NPC. We will perform a systematic and in-depth investigation of the molecular interactions between actin and the NPC using pharmacological and genetic approaches in iPSC-derived neurons carrying the C9ORF72 mutation. 2) Identify the consequences of NCT defects on the nuclear and cytoplasmic proteome in response to cellular stimulation. By combining an “omics” approach with a candidate approach (i.e. CREB pathway), we will analyze changes in the nuclear and cytoplasmic proteome in iPSC-derived neuronal models of ALS/FTD following stimulations, and we will test the potentials of cytoskeleton modulation to rescue such defects. 3) Define how defects in nuclear import affect the transcriptional response to cellular stimulation. We will analyze changes in gene expression and splicing profile in ALS/FTD neurons following cell stimulation by RNA-Seq. Network analysis performed by integrating the results from the transcriptomic and proteomics approaches will identify ALS/FTD relevant altered pathways. Modulation of the cytoskeleton will be used to rescue the identified defects. At the completion of this research, we will have identified the molecular and cellular mechanisms that control the stability and function of the NPC, and the functional consequences that the disruption of NCT has on the resilience of neurons in ALS/FTD. Gaining insights on what pathways upstream and downstream of the NPC are affected in ALS/FTD will greatly expand our understanding of disease pathogenesis and will allow us to identify yet unexplored avenues for therapy in these diseases. Ultimately, we expect that our research will lead to more effective therapeutic strategies that take advantage of the crosstalk between NPC, RNA regulation, and cytoskeleton. This research will have a broad impact on a spectrum of diseases including but not limited to ALS/FTD.