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

研究 NEK1 激酶中 ALS/FTD 相关突变对疾病病理生理学的贡献

基本信息

批准号：
10753020
负责人：
Evangelos Kiskinis
金额：
$ 76.6万
依托单位：
NORTHWESTERN UNIVERSITY AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-07-15 至 2028-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10753020
关键词：
ALS patients Amyotrophic Lateral Sclerosis Antibodies Autopsy Behavior Brain C9ORF72 CRISPR/Cas technology Cell Cycle Regulation Cell Line Cell model Cell physiology Cessation of life Clinical Cytoskeleton DNA Damage Defect Disease Drosophila genus Engineering Exhibits Family Frontotemporal Dementia Functional disorder Genes Genetic Genetic Heterogeneity Genetic study Homeostasis Homologous Gene Human Impairment In Vitro Induced pluripotent stem cell derived neurons Language Lead Light Link Longevity Loss of Heterozygosity Mass Spectrum Analysis Mediating Microtubule Proteins Microtubules Modeling Motor Motor Neurons Movement Muscle Mutagenesis Mutation NIMA Nature Nerve Degeneration Nervous System Neurodegenerative Disorders Neuronal Dysfunction Neurons Nuclear Import Paralysed Pathogenesis Pathway interactions Patients Personality Phosphorylation Phosphotransferases Physiological Play Predisposition Proteins Proteomics RNA Interference RNA metabolism Resources Role Small Interfering RNA Spinal Cord System Temporal Lobe Testing Tissues Toxic effect Translating Variant Work age related causal variant comorbidity differential expression effective therapy exome sequencing experimental study fly frontal lobe frontotemporal lobar dementia amyotrophic lateral sclerosis genetic variant in vivo in vivo Model induced pluripotent stem cell loss of function mimetics motor disorder motor impairment motor neuron function mutant neuropathology neurotoxicity novel phosphoproteomics protein expression proteostasis receptor response superoxide dismutase 1 therapeutic candidate therapeutic target

项目摘要

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.

肌萎缩侧索硬化症（ALS）是一种破坏性的神经退行性疾病，其特征是逐渐无法控制肌肉运动。 ALS 患者常合并额颞叶病变痴呆症 (FTD)，也称为 ALS/FTD。 ALS 的临床表现是由选择性介导的连接中枢神经系统和肌肉组织的上下运动神经元 (MN) 功能障碍和退化。绝大多数 ALS 本质上是散发性的，而 10% 的患者患有家族性 ALS 疾病，这使得能够识别致病基因变异。 ALS 可能是由突变引起的编码涉及多种细胞功能的蛋白质的基因，包括 RNA 代谢、蛋白质稳态和细胞骨架稳态。最近的遗传学研究强调 NIMA 相关激酶 1 (NEK1) 是一个主要的 ALS 的遗传因素。 NEK1 功能丧失的遗传变异使许多人对 ALS 易感占所有病例的 2%。 NEK1 在 CNS 中的具体作用和功能仍未解决。还剩下什么导致 NEK1 ALS 病理生理学突变的细胞机制尚不清楚。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 MN、体内果蝇模型和 ALS-NEK1 死后患者 CNS 组织，以：a) 确定 ALS 相关的机制突变损害 MN 功能； b) 表征 NEK1 依赖性磷酸化的生理底物； c) 验证这些变化对 ALS 神经病理学的贡献。我们将检验假设 NEK1 突变通过破坏激酶对细胞通路的调节作用而导致神经毒性对于 MN 功能至关重要。在初步实验中，我们发现 NEK1 缺陷的 iPSC 衍生的 MN 表现出微管 (MT) 动力学破坏和核输入受损。在目标 1 中，我们将确定是否这些缺陷与 ALS 相关 NEK1 的大量无义和错义相关变体。在初步实验中，我们发现 NEK1 相互作用子在 MT 中的功能得到丰富细胞骨架和核输入以及 NEK1 水平的降低导致蛋白质的差异表达参与这些途径。在目标 2 中，我们将确定 NEK1 磷酸化的生理底物通过进行磷酸化蛋白质组质谱分析并探究 MN 的功能效应差异磷酸化。在初步实验中，我们确定 Niki 是最接近的果蝇同源物 NEK1 并使用 RNAi 系，我们发现它对于运动功能和生存至关重要。在目标 3 中，我们将确定 NEK1 在果蝇完整神经系统中的功能，并验证我们的研究结果体内细胞模型。我们的研究将揭示受到以下因素损害的细胞机制神经元中的突变 NEK1 可能会发现很大一部分神经元的潜在治疗靶点 ALS/FTD 患者。