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.

肌萎缩性侧索硬化症（ALS）是一种毁灭性的神经退行性疾病，其特征是渐进的无法控制肌肉运动。 ALS患者通常与额颞痴呆症（FTD），也称为ALS/FTD。 ALS的临床表现是由选择性介导的将CNS连接到肌肉组织的上和下运动神经元（MN）的功能障碍和变性。绝大多数ALS本质上都是零星的，而10％的患者患有家族形式疾病，已经鉴定出病因遗传变异。 ALS可能是由突变引起的在编码参与RNA代谢的各种细胞功能的蛋白质的基因中和细胞骨架稳态。最近的遗传研究突出了NIMA相关的激酶1（NEK1）作为主要的对ALS的遗传贡献者。 NEK1的功能丧失遗传变异赋予ALS的易感性多数所有情况中有2％。 NEK1在中枢神经系统中的特定作用和功能仍未解决。还有什么难以捉摸的是导致突变NEK1 ALS病理生理学的细胞机制。在本研究中，我们将使用NEK1细胞模型，诱导多能干细胞（IPSC）患者衍生的MN，在体内果蝇模型和ALS-NEK1验尸患者CNS组织至：a）确定与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突变通过破坏激酶在细胞途径上的调节作用而引起神经毒性对于MN功能至关重要。在初步实验中，我们发现NEK1缺陷型IPSC衍生的MNS 表现出破坏的微管（MT）动力学和核进口受损。在AIM 1中，我们将确定是否这些缺陷在一系列无义和错过ALS相关的NEK1的背景下是相关的变体。在初步实验中，我们发现NEK1相互作用者在MT中的功能丰富细胞骨架和核进口以及NEK1水平的降低导致蛋白质的差异表达参与这些途径。在AIM 2中，我们将确定NEK1磷酸化的生理底物在MN中，通过进行磷蛋白质组学质谱分析并询问的功能效应差异磷酸化。在初步实验中，我们将尼基确定为最接近的果蝇同源物 NEK1和使用RNAi线的含量，我们发现它对于运动功能和存活至关重要。在目标3中，我们将确定NEK1在完整的苍蝇神经系统中的功能，并验证我们对体内细胞模型。我们的研究将阐明被损害的细胞机制神经元中的突变nek1，可能会发现大量的潜在治疗靶标 ALS/FTD患者。