Project Summary/Abstract The prevalence of Alzheimer’s disease (AD), a progressive neurodegenerative disease, continues to increase at a staggering rate. Current AD therapies provide modest symptomatic relief, creating a need for disease- modifying therapies, which requires a better understanding of the mechanisms underlying AD. AD is characterized by the spread of tau pathology through the brain in a stereotypical pattern that correlates well with the disease progression. However, a critical need still exists to understand the molecular underpinnings of tau toxicity in AD and thereby identify promising therapeutic targets to slow and/or halt AD progression. We previously showed that tau aggregates disrupt anterograde fast axonal transport in the squid giant axon by activating the protein phosphatase 1 (PP1)- glycogen synthase kinase 3β (GSK3β) signaling pathway which causes the release of cargo from the anterograde motor protein kinesin through phosphorylation of kinesin light chains by GSK3β. In tau aggregates, the exposure of an N-terminal epitope called the phosphatase activating domain (PAD) activates this signaling pathway via interaction with and activation of PP1 in the squid giant axon. Overactivity of this pathway results in aberrant cargo release and thus axonal transport deficits in the squid. Furthermore, we recently demonstrated that interactions between the mutant P301L tau and PP1 results in disrupted axonal transport in mammalian primary neurons, and the pathology is rescued upon PP1γ knockdown. PAD exposure and axonal degeneration are evident early in the progression of AD and in other tauopathies. Therefore, understanding the potentially toxic effects of PAD exposure is directly relevant to disease. Mounting evidence suggests that tau pathology is propagated throughout the brain potentially in a prion-like process involving cell-to-cell tau seeding. However, the functional consequences of seeded aggregation of endogenous tau is not fully understood. The proposed experiments are designed to test the hypothesis that AD brain-derived tau aggregates induce axonal degeneration in tau-seeded mammalian neurons via activation of the PAD-PP1-GSK3β signaling pathway. The pathological consequences of seeding with AD-derived tau pre-formed fibrils (PFF-tau) will be determined in functional live-cell axonal transport assays. The activity of PP1 and GSK3β in PFF-tau-treated neurons will be measured using biochemical assays. Furthermore, immunofluorescent staining, confocal microscopy and stereological measurement techniques will be used to assess axonal and synaptic degeneration. Upon completion of this project, we will have a better understanding of whether tau seeding in mammalian primary neurons causes toxicity through activation of the PAD-PP1-GSK3β pathway. This new knowledge could inform the development of more effective therapies for AD.