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

项目总结 C9orf72连锁ALS/FTD中作为重复相关非AUG翻译修饰物的代谢扰动 发生在C9orf72(C9)基因中的-(GGGGGCC)n-六核苷酸重复序列扩展(HRE)是最常见的原因 并通过非规范机制导致神经毒性二肽重复蛋白(Dprs)的产生。 称为重复关联非8月(RAN)转换。通过多个因素激活综合应激反应 不同的细胞应激源已经被证明增加RAN翻译的发生而不增加典型的 8月驱动的翻译。ISR激活的核心事件是真核起始的α亚单位的磷酸化 因子2(eIF2α)通过以下四种途径之一：PERK、PKR、GCN2和HRI，每种途径都可以被不同的细胞激活 压力源。我建议解决代谢扰动的作用及其对C9连锁范围的潜在调节作用 翻译。这可以通过激活ISR来实现，特别是通过GCN2和PERK途径，这两条途径是 分别被氨基酸剥夺和内质网应激激活。这对于理解C9-ALS/FTD特别相关 致病机制是因为多种代谢紊乱，包括高代谢、葡萄糖/能量 在肌萎缩侧索硬化症中观察到线粒体呼吸链的缺陷和改变，并可能导致ISR激活。为 例如，神经元中的葡萄糖缺乏可能迫使细胞代谢氨基酸以获得能量，从而潜在地导致葡萄糖缺乏 氨基酸(A.A.)通过GCN2供应和激活ISR。同样，呼吸链的扰动可能会导致 过量的ROS形成，导致内质网应激，并通过PERK激活ISR。我建议测试假设，在C9中- ALS/FTD，疾病驱动的能量失衡通过ISR激活促进RAN翻译。我会用试管和试管婴儿 疾病的体内模型，包括从C9患者诱导的大脑皮层和运动神经元分化 多能干细胞(C9-IPSCs)以及C9orf72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 ISR激活和RAN翻译使用生化、分子和细胞生物学技术以及几种行为 分析。

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.