Project Summary - Despite decades of research into Alzheimer's disease (AD), disease-modifying treatments for AD remain elusive. This is significantly due to challenges in understanding the molecular and structural basis of AD. One of the two hallmarks of AD is the neurofibrillary tangles formed by the intrinsically disordered microtubule (MT)-associated protein tau. Spreading of tau filaments in the brain is the basis of neuropathological staging of AD. AD tau is hyperphosphorylated, truncated, and decorated with other posttranslational modifications (PTMs). However, how these PTMs cause tau to dissociate from MTs and misfold into -sheet amyloids, and how tau crosses the lipid membrane to spread its pathology, is not known. AD paired helical filament (PHF) tau fibrils have a C-shaped -sheet core that encompasses part of the MT-binding repeats. But the majority of the protein, which contains most of the disease-relevant PTMs, is too disordered to be seen in cryo-electron microscopy data. Here we propose to employ solid-state NMR (ssNMR) spectroscopy, electron microscopy, mouse neuron toxicity assays, and other biochemical approaches to understand the molecular structures and dynamics of AD tau filaments, membrane-bound tau, and MT-bound tau. We hypothesize that specific charge-charge interactions underlie the varying conformations, dynamics and properties of tau when self-aggregated and when bound to its cellular partners. In the last four years, we demonstrated the feasibility of applying ssNMR to study the structures and dynamics of full-length tau fibrils formed in vitro and seeded by AD PHF tau. We will now apply this expertise to answer three questions. In Aim 1, we will investigate how phosphorylation and truncation cause AD PHF tau by determining the structures of phosphorylated tau (p-tau) fibrilized without anionic cofactors; searching for minimum constructs that replicate the AD PHF tau structure and properties; and characterizing the dynamic structures of the semi-mobile proline-rich region of tau. In Aim 2, we will investigate tau interactions with lipid membranes by measuring the conformation, dynamics and membrane insertion of monomeric tau bound to small and large unilamellar vesicles. We will determine the structures of membrane-induced tau aggregates, and probe how phosphorylation and truncation affect the structure and dynamics of membrane-bound tau. These experiments should shine light on how lipid membranes nucleate tau aggregates and how aggregated tau crosses the membrane. In Aim 3, we will investigate the structures of MT-bound tau as a function of phosphorylation, and probe how arginine-phosphate interactions in the R' domain affect tau binding to MTs. A joint study of the fibrillar, membrane-bound and MT-bound tau is crucial for understanding how tau converts from its intrinsically disordered structure to an aggregated structure that propagates in a prion-like manner. This understanding should inform the future design o...