PROJECT SUMMARY/ABSTRACT Protein aggregation is a hallmark of neurodegenerative diseases, including Alzheimer’s, and these diseases lack effective therapeutics. Moreover, we lack an understanding of the molecular and cellular mechanisms controlling protein aggregation in the human brain, which would enable new therapeutic strategies. The protein tau is the major constituent of aggregates in the brain in a number of neurodegenerative diseases, collectively called tauopathies, including Alzheimer’s Disease. Although a number of disease- associated tau mutations are known, we lack a comprehensive understanding of how sequence controls tau aggregation and misfolding. Furthermore, tau, despite being widely expressed in the brain, aggregates at disease-onset in only specific neuronal subtypes. This phenomenon, called selective vulnerability, suggests that differential expression of cellular factors plays a key role in tau misfolding and aggregation. I hypothesize that cellular factors and sequence changes control specific tau states that determine its aggregation and misfolding in human neurons. Here, I propose two systematic, unbiased strategies, CRISPR-based functional genomics and Deep Mutational Scanning (DMS), to comprehensively dissect how both sequence determinants and cellular factors control tau misfolding and aggregation in iPSC-derived neurons. Integration of results from these studies with biophysical and structural follow-up experiments will determine specific tau states that are essential for its aggregation. By performing systematic studies in human neurons, I am uniquely positioned to determine classes of tau mutations and cellular factors that underlie tau aggregation and misfolding. I have performed a focused proof-of-principle functional genomics screen and identified the peptidyl- proline isomerases (PPIases) FKBP1A and PPIH as regulators of tau oligomerization. In Aim 1, I will characterize how the PPIases FKBP1A and PPIH control tau misfolding and aggregation in collaboration with my co-sponsor Dr. Bill DeGrado and collaborator Dr. Lukasz Joachimiak. Aim 2 uses a functional genomics approach to identify cellular factors that control tau aggregation and misfolding. Follow-up studies will determine how identified factors control tau conformations on-pathway to aggregation. Aim 3 uses DMS to comprehensively determine the sequence factors that control tau aggregation and misfolding, with a specific emphasis on dissecting the role of PTMs. Completion of these aims will result in a comprehensive understanding of the underlying cellular factors and sequence determinants that control tau misfolding and aggregation, and reveal potential therapeutic targets.