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.

PROJECT NARRATIVE A mutation in the C9orf72 gene is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two devastating neurodegenerative diseases which lead to rapid loss of motor and/or cognitive function. Both diseases are incurable and lead to death usually within 3-5 years of symptom onset, highlighting the need for a more comprehensive understanding of disease pathogenesis to develop new therapies. The proposed research will include a comprehensive assessment of potential disease modifiers using multiple disease-relevant model systems, which will advance the field’s understanding of the molecular underpinnings of these diseases and facilitate the discovery of novel therapeutic targets.