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

项目摘要 导致ALS/FTD中神经元死亡的分子缺陷的详细见解仍然缺失，阻碍了研究的进展。 发展治愈。核孔复合体（NPC）和核质转运（NCT）缺陷 已被提出在ALS/FTD病理学中发挥核心作用。一个突出的知识差距是什么， NCT受损对神经元弹性的机制、后果和致病相关性， 神经退行性疾病和衰老期间。本申请的目的是确定在机械 使功能失调并导致NCT失败的新细胞和分子途径水平化 最终导致神经退化特别是，我们将测试的假设，肌动蛋白细胞骨架是一个 核稳定性和NPC功能的主要调节剂，ALS/FTD中该途径的变化导致 神经元科普压力和生理刺激的能力减弱。 本提案的具体目标是：1）定义肌动蛋白如何调节细胞的功能和功能障碍， 届全国人大我们将对肌动蛋白和细胞膜之间的分子相互作用进行系统而深入的研究。 在携带C9 ORF 72的iPSC衍生的神经元中使用药理学和遗传学方法的NPC 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衍生的神经元模型中细胞核和细胞质蛋白质组的变化 的ALS/FTD的刺激后，我们将测试的潜力，细胞骨架调制，以挽救这种 缺陷3)定义核输入缺陷如何影响细胞转录反应， 刺激.我们将分析ALS/FTD神经元中基因表达和剪接谱的变化， 通过RNA-Seq.通过整合来自转录组学的结果进行网络分析， 蛋白质组学方法将确定ALS/FTD相关的改变途径。细胞骨架的调节将是 用于挽救已识别的缺陷。 在这项研究完成后，我们将确定控制的分子和细胞机制， NPC的稳定性和功能，以及NCT的破坏对NCT的功能影响。 ALS/FTD中神经元的弹性。深入了解NPC的上游和下游途径 将极大地扩展我们对疾病发病机制的理解，并使我们能够 确定尚未探索的治疗这些疾病的途径。最终，我们希望我们的研究能够 更有效的治疗策略，利用NPC，RNA调节， 和细胞骨架。这项研究将对一系列疾病产生广泛的影响，包括但不限于 ALS/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.