The recent identification of mutations in the C9orf72 and TDP-43 (TARDBP) genes causative of familial forms of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) has uncovered a new reality of a continuous FTD/ALS spectrum. Non-coding C9orf72-GGGGCC repeats produce toxic dipeptides by RAN translation, making mutant C9orf72 a proteinopathy, like TDP-43. Additionally, nucleo-cytoplasmic shuttling seems to underlie both pathologies, hinting at robust molecular connections. Despite these recent advances, we still have an incomplete understanding of the gene networks disrupted by mutant TDP-43 and C9orf72 and how these two distinct triggers induce overlapping pathologies. A comprehensive, genome-wide genetic screen will help uncover globally the molecular networks mediating the pathogenesis of TDP-43 and C9orf72. Our long-term goal is to identify novel and effective therapeutic targets for TDP-43- and C9orf72-based pathologies. The overall objectives of this proposal are to (1) characterize the gene networks underlying the neurotoxicity of TDP-43M337V and the PR-36 dipeptide in Drosophila, and (2) identify the best targets for therapeutic intervention. Our HYPOTHESIS is that the gene networks disrupted by TDP-43M337V and PR-36 will identify a few common pathways AND several trigger-specific mechanisms representing the wide spectrum of FTD and ALS. This hypothesis is based on preliminary results from a genetic screen of 6,600 RNAi alleles searching for modifiers of TDP-43M337V and PR-36 neurotoxicity in the Drosophila eye. So far, we have verified 55 TDP-43 suppressors, 46 PR-36 suppressors, 12 common suppressors, and 150 enhancers for each. Among the suppressors, we confirmed the role of genes involved in nuclear transport. We also found several suppressors involved in mRNA synthesis and splicing, cell signaling, proteostasis, vesicle trafficking, and others suggesting new pathogenic mechanisms. Due to the preliminary nature of these results, this R21 will support the next several steps to validate the modifiers, create the gene networks for each model, and logically rank the candidates (Aim 1). Then, we will test the protective activity of the top five suppressors in the adult brain under therapeutic conditions (Aim 2). The rationale for this proposal is that LOF mutations identifying the gene networks mediating TDP-43 and PR-36 proteotoxicity will uncover novel therapeutic targets for FTD/ALS with high therapeutic potential. The proposed experiments are SIGNIFICANT because they are expected to (1) increase our knowledge of the pathogenic mechanisms implicated in FTD/ALS and (2) identify novel therapeutic targets for FTD/ALS. The INNOVATIONS of this proposal are (1) the dual screen, (2) the genome-wide LOF screen, (3) the gene network analysis to rank the candidates, and (4) the inducible fly models to test the activity of candidate genes under therapeutic conditions. This project is appropriate for an R21 because of the preliminary, risky nature of the genetic screen and the high-reward potential of finding common therapeutic targets for TDP-43 and PR-36.