This is a proposal to combine our three NINDS funded R01 projects into one larger R35 award, enabling us the time and flexibility to explore important biological questions relevant to human neurodegenerative diseases. Over the past eight years, my laboratory has used a combination of yeast and human genetics to define novel mechanisms of ALS, FTD, and Parkinson's disease. These experiments have led to the discovery of ataxin 2 intermediate-length polyglutamine expansions as a major genetic risk factor for ALS, the discovery of RNA lariat debranching enzyme as a powerful therapeutic target for TDP-43 proteinopathies, a new cellular pathway to explain how C9orf72 mutations could cause neurodegeneration and unexpected connections between seemingly distinct Parkinson's disease genes. In preliminary studies, we have found that genetic reduction of ataxin 2 in mouse profoundly extends survival of TDP-43 transgenic mice (>80% increase in lifespan). We propose studies to explore how ataxin 2 protects against TDP-43 proteinopathy, test ataxin 2 in other mouse models (e.g., C9orf72 and FUS/TLS), and to pursue antisense oligonucleotides targeting ataxin 2 in human cell models. While our previous work has stemmed from yeast models and genetic modifier screens, we now propose an ambitious advance – performing genomewide modifier screens in human cells using CRISPR/Cas9 gene activation and inactivation libraries. We have already performed three pilot screens with C9orf72 models and have identified several potent modifiers, which we will validate in primary neurons and mouse models. I plan to take my lab into this new direction by performing CRISPR screens in human cells with TDP-43, FUS, alpha-synuclein, ataxin 2, and further C9orf72 models (e.g., RNA vs. DPRs). We are also interested in the process of RAN (repeat-associated non-ATG) translation, which has emerged as a powerful facet of several nucleotide-repeat diseases (including c9ALS/FTD). We propose experiments to discover the molecular mechanisms of RAN translation in order to design specific inhibitors and we have already identified at least two genes that seem to be required for RAN translation. Together, we present an ambitious research program aimed at defining novel mechanisms of human neurodegenerative diseases and then intensely working to translate those mechanisms to novel therapies to help treat these devastating conditions.