Investigating the Contribution of ALS/FTD-Associated Mutations in the NEK1 Kinase to Disease Pathophysiology

肌萎缩侧索硬化症(ALS)是一种毁灭性的神经退行性疾病，其特征是 逐渐丧失控制肌肉运动的能力。肌萎缩侧索硬化症患者常合并额颞部 痴呆症(FTD)，也称为ALS/FTD。肌萎缩侧索硬化症的临床表现由选择性 连接中枢神经系统和肌肉系统的上下运动神经元(MN)功能障碍和变性。 绝大多数肌萎缩侧索硬化症是散发性的，而10%的患者患有家族性形式的肌萎缩侧索硬化症。 疾病，这使得鉴定致病基因变异成为可能。肌萎缩侧索硬化症可由突变引起 在编码涉及多种细胞功能的蛋白质的基因中，从RNA新陈代谢、蛋白平衡 和细胞骨架的动态平衡。最近的遗传学研究强调了NIMA相关激酶1(NEK1)是一种主要的 肌萎缩侧索硬化症的遗传因素。NEK1功能丧失的遗传变异使多达20%的人对ALS易感性 占所有病例的2%。NEK1在中枢神经系统中的具体作用和功能仍未解决。也留下了什么 导致突变的NEK1 ALS病理生理学的细胞机制是难以捉摸的。在本研究中，我们将 使用NEK1细胞模型、诱导多能干细胞(IPSC)患者来源的MNS、体内果蝇模型 和ALS-NEK1死后患者CNS组织：a)确定ALS相关的机制 突变损害MN功能；b)表征NEK1依赖的磷酸化的生理底物； 以及，c)验证这些变化对ALS神经病理学的贡献。我们将检验这一假设 NEK1突变通过扰乱细胞通路上激酶的调节作用而导致神经毒性 是MN功能所必需的。在初步实验中，我们发现NEK1缺陷的IPSC来源的MNS 表现出微管(MT)动力学紊乱和核进口受损。在目标1中，我们将确定 这些缺陷与一系列与ALS相关的无稽之谈和错义NEK1相关 变种。在初步实验中，我们发现NEK1相互作用在MT中的功能得到了丰富 细胞骨架和核输入以及NEK1水平降低导致蛋白质差异表达 参与了这些途径。在目标2中，我们将确定NEK1磷酸化的生理底物 在MNS中通过进行磷酸蛋白质组质谱分析和询问功能效应 差异磷酸化。在初步实验中，我们确定Niki是与果蝇最接近的同源物 通过使用RNAi细胞系，我们发现NEK1对运动功能和生存是必不可少的。在《目标3》中，我们将 确定NEK1在果蝇完整神经系统中的功能，并验证我们的发现对 体内的细胞模型。我们的研究将有助于揭示 突变的NEK1在神经元中，并可能发现相当大比例的 ALS/FTD患者。

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that is characterized by a progressive inability to control muscle movement. ALS patients are often comorbid with frontotemporal dementia (FTD), also known as ALS/FTD. The clinical manifestation of ALS is mediated by the selective dysfunction and degeneration of upper and lower motor neurons (MNs) that connect the CNS to the musculature. The overwhelming majority of ALS is sporadic in nature, while 10% of patients suffer from familial forms of disease, which have enabled the identification of causative genetic variants. ALS can be caused by mutations in genes that encode proteins involved in diverse cellular functions ranging from RNA metabolism, proteostasis and cytoskeletal homeostasis. Recent genetic studies have highlighted NIMA-related kinase 1 (NEK1) as a major genetic contributor to ALS. Loss-of-function genetic variants in NEK1 confer susceptibility to ALS in as many as 2% of all cases. The specific role and function of NEK1 in the CNS remains unresolved. What also remains elusive is the cellular mechanisms that lead to mutant NEK1 ALS pathophysiology. In the present study, we will use NEK1 cellular models, induced pluripotent stem cell (iPSC) patient-derived MNs, in vivo Drosophila models and ALS-NEK1 postmortem patient CNS tissue to: a) determine the mechanisms by which ALS-associated mutations impair MN function; b) characterize the physiological substrates for NEK1-dependent phosphorylation; and, c) validate the contribution of these changes towards neuropathology in ALS. We will test the hypothesis that NEK1 mutations cause neurotoxicity by disrupting the regulatory role of the kinase on cellular pathways that are essential for MN function. In preliminary experiments, we found that NEK1-deficient iPSC-derived MNs exhibit disrupted microtubule (MT) dynamics and impaired nuclear import. In Aim 1 we will determine whether these defects are relevant in the context of an extensive set of nonsense and missense ALS-associated NEK1 variants. In preliminary experiments, we found that NEK1 interactors are enriched for function in the MT cytoskeleton and nuclear import and that reduction of NEK1 levels results in differential expression of proteins involved in these pathways. In Aim 2 we will determine the physiological substrates for NEK1 phosphorylation in MNs by conducting phosphoproteomic mass spectrometry analysis and interrogating the functional effects of differential phosphorylation. In preliminary experiments we identified Niki as the closest Drosophila homologue of NEK1 and using RNAi lines we found that it is essential for motor function and survival. In Aim 3 we will determine the function of NEK1 in the intact nervous system of flies and validate our findings on the effects of the cellular models in vivo. Our studies will shed light into the cellular mechanisms that are compromised by mutant NEK1 in neurons and will likely uncover potential therapeutic targets for a significant percentage of ALS/FTD patients.