Project Summary Pathologic Tau protein self-assembly templating and stereotypical regional spreading along neuronal networks correlates with loss of neurons and is central to clinical progression and staging in Alzheimer’s disease (AD). In diseased brain, Tau molecules begin to form filamentous aggregates with a rigid β-sheet rich core. Although some Tau protein fragments can spontaneously aggregate, recombinant full-length Tau produced in vitro or when expressed in mammalian cells does not aggregate spontaneously even at supersaturation. To initiate Tau protein to form aggregates, some unknown energy barrier must be overcome. Research shows that inducing Tau monomer to form a nucleus leading to filamentous elongation can be a heterogenous association such as with a membrane, small molecules, or peptides stabilizing the misfolded species for subsequent elongation steps. Proteolysis is a process of protein degradation which also controls multiple cellular functions. Tau is a natively unfolded protein sensitive to protease digestion and the longest human brain isoform contains more than forty cleavage sites which leads to hundreds of possible Tau-derived peptides. Peptides have been shown to rapidly induce the misfolding and aggregation of tau in vitro and in mammalian cells. Here we propose to test and rank the propensity of Tau aggregation induction by each protease-cleaved Tau peptide after incubation with full-length recombinant Tau isoforms and subsequently capture aggregate gross morphology using transmission electron microscopy (TEM). Intracellular signaling activity by Tau peptides will be detected in cell culture to further characterize each peptide. Peptides inducing full-length Tau protein to aggregate or having active cellular signaling properties may implicate protease components dysregulated in disease. Because many known cleavage sites on Tau protein remain uncharacterized, this study aims to collect preliminary data for future funding and will begin groundwork for long-term research of proteolytic post- translational modification to identify those sites on Tau which may produce toxic peptides in neurons.