Metabolic perturbations as a modifier of repeat-associated non-AUG translation in C9orf72-linked ALS/FTD

代谢扰动作为 C9orf72 相关 ALS/FTD 中重复相关非 AUG 翻译的修饰剂

• 批准号: 10271263
• 负责人: Andrew Nelson
• 金额: $ 4.6万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-17 至 2022-09-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10271263
• 关键词: Address; Amino Acids; Amyotrophic Lateral Sclerosis; Astrocytes; Autopsy; Bacterial Artificial Chromosomes; Behavioral; Biochemical; Biological Models; Blood - brain barrier anatomy; Brain; C9ORF72; Cell model; Cells; Cellular Metabolic Process; Cellular biology; Cessation of life; Chronic; Cognitive; Detection; Dipeptides; Disease; Disease model; Energy Metabolism; Energy-Generating Resources; Engineering; Enzyme-Linked Immunosorbent Assay; Eukaryotic Initiation Factor-2; Event; Exhibits; Genes; Genetic; Glucose; Goals; Human; Immunofluorescence Immunologic; Impairment; In Vitro; Induced pluripotent stem cell derived neurons; Initiator Codon; Insulin; Intervention; Introns; Knowledge; Lead; Link; Mediating; Metabolic; Metabolic Pathway; Mitochondria; Modeling; Molecular; Molecular Biology; Motor; Motor Neurons; Mus; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurons; PERK kinase; Pathogenesis; Pathogenicity; Pathology; Pathway interactions; Patients; Pharmacology; Phenotype; Phosphorylation; Production; Proteins; Reporter; Reporting; Research; Respiratory Chain; Role; SLC2A1 gene; Spinal Cord; Symptoms; Techniques; Testing; Tissues; Transgenic Mice; Transgenic Organisms; Translating; Translations; Viral Vector; base; biological adaptation to stress; cognitive function; deprivation; detection method; disease mechanisms study; disease phenotype; endoplasmic reticulum stress; energy balance; frontotemporal lobar dementia-amyotrophic lateral sclerosis; glucose metabolism; glucose transport; immunocytochemistry; in vivo; in vivo Model; induced pluripotent stem cell; interest; knock-down; live cell imaging; mortality risk; mouse model; neuron loss; neurotoxic; new therapeutic target; novel therapeutics; nutrient metabolism; oxidative damage; protein degradation; respiratory; solute; stressor; tool

PROJECT SUMMARY Metabolic perturbations as a modifier of repeat-associated non-AUG translation in C9orf72-linked ALS/FTD The -(GGGGCC)n- hexanucleotide repeat expansion (HRE) occurring in the C9orf72 (C9) gene is the most common cause of ALS and FTD and leads to the production of neurotoxic dipeptide repeat proteins (DPRs) by a noncanonical mechanism known as repeat-associated non-AUG (RAN) translation. Activation of the integrated stress response by a number of different cellular stressors has been shown to increase the occurrence of RAN translation without increasing the canonical AUG-driven translation. The core event in the ISR activation is phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2α) by one of four kinases: PERK, PKR, GCN2, and HRI, each of which can be activated by different cellular stressors. I propose to address the role of metabolic perturbations and their potential modifier effect on C9-linked RAN translation. This could occur via activation of the ISR, specifically through the GCN2 and PERK pathways, which are activated by amino acid deprivation and ER stress, respectively. This is particularly relevant to understanding C9-ALS/FTD pathogenic mechanisms because a variety of metabolic perturbations, including hypermetabolism, glucose/energy deficiency, and alterations to the mitochondrial respiratory chain, are observed in ALS and could lead to ISR activation. For instance, glucose deficiency in neurons could force cells to metabolize amino acids for energy, potentially causing a deficit in amino acid (a.a.) supply and activating the ISR via GCN2. Likewise, perturbations to the respiratory chain could cause excessive ROS formation, leading to ER stress and activating the ISR via PERK. I propose to test the hypothesis that in C9- ALS/FTD, disease-driven energy imbalances promote RAN translation via ISR activation. I will employ both in vitro and in vivo models of the disease, including cortical and motor neurons differentiated from C9 patient-derived induced pluripotent stem cells (C9-iPSCs) as well as a C9orf72 bacterial artificial chromosome (BAC) transgenic mouse model. I will perturb the energy balance in these models by targeting critical metabolic pathways. Then I will evaluate the impact on ISR activation and RAN translation using biochemical, molecular and cell biology techniques and several behavioral analyses.

项目摘要 代谢紊乱作为C9 orf 72-连锁ALS/FTD中重复序列相关非AUG翻译的调节剂 C9 orf 72（C9）基因中的-（GGGGCC）n-六核苷酸重复扩增（HRE）是最常见的原因 的ALS和FTD，并导致产生神经毒性二肽重复蛋白（DPR）的非经典机制 称为重复相关非AUG（RAN）翻译。激活的综合应激反应，由一些 不同的细胞应激源已被证明增加RAN翻译的发生，而不增加典型的细胞应激。 AUG驱动的翻译。ISR激活的核心事件是真核生物启动子α亚基的磷酸化， EIF 2 α是由四种激酶之一激活的：PERK，PKR，GCN 2和HRI，每一种都可以被不同的细胞因子激活。 压力源我建议解决的作用，代谢扰动和其潜在的修饰剂的影响，对C9连接的RAN 翻译.这可能通过ISR的激活发生，特别是通过GCN 2和PERK途径， 分别由氨基酸剥夺和ER应激激活。这与理解C9-ALS/FTD特别相关 致病机制，因为各种代谢紊乱，包括高代谢，葡萄糖/能量 缺乏和线粒体呼吸链的改变，在ALS中观察到，并可能导致ISR激活。为 例如，神经元中的葡萄糖缺乏会迫使细胞代谢氨基酸以获得能量，从而可能导致缺陷 氨基酸（a.a.）通过GCN 2提供并激活ISR。同样，呼吸链的扰动可能导致 过量的ROS形成，导致ER应激并通过PERK激活ISR。我建议测试假设，在C9- ALS/FTD，疾病驱动的能量失衡通过ISR激活促进RAN翻译。我将在体外和 在疾病的体内模型中，包括从C9患者衍生的诱导分化的皮质和运动神经元。 多能干细胞（C9-iPSC）以及C9 orf 72细菌人工染色体（BAC）转基因小鼠模型。我 将通过靶向关键的代谢途径来扰乱这些模型中的能量平衡。然后我将评估对 使用生物化学、分子和细胞生物学技术和几种行为生物学方法， 分析。