PROJECT SUMMARY/ABSTRACT In the US, Lewy body dementias (LBD) and Alzheimer’s disease (AD) affect over 7 million people. No treatment slows their inexorable decline. Broad evidence implicates malicious roles of α-synuclein (ASyn), tau and Abeta (A) proteins in these dementias. LBD and AD are characterized by the sequential misfolding of ASyn, tau and A into toxic oligomers and fibrils that propagate in prion-like fashion and accumulate in pathological deposits, with ASyn the predominating pathology in LBD and A and tau pathologies predominating in AD. ASyn or A precursor gene alterations cause LBD and AD respectively. Although central to disease, ASyn, A and tau mis- folding have been challenging to target therapeutically. ASyn, tau and A pathologies correlate with LBD and AD decline. Elevated levels of the stress-induced chaperone Hsp70 protects against ASyn misfolding and neu- rodegeneration in LBD cell and animal models. However, there is minimal mechanistic understanding of this important protective pathway. In particular, it is generally assumed that the canonical and promiscuous mode of Hsp70 action underlies Hsp70-ASyn protection. Consequently, translational investigations have been stymied by the anticipation of undesirable side effects of therapeutically targeting Hsp70 canonical actions. Our data contradict this assumption, providing a potentially more targeted and exploitable mechanism of Hsp70-mediated protection in disease. We find that Hsp70 blocks the earliest and most neurotoxic stage of ASyn misfolding, ASyn oligomerization, by interacting at a previously unknown site separate from its canonical site of action. It is therefore plausible that targeting this non-canonical mechanism would protect against disease. Importantly, this approach holds the potential to correct ASyn misfolding without disrupting critical cellular processes. Our central hypothesis is that this novel, non-canonical blockage of ASyn oligomerization by Hsp70 is protective in LBD and AD. The next steps to support and exploit this hypothesis are to determine the molecular mechanism of Hsp70’s engagement with ASyn, to validate this mechanism as protective, and test its action on additional AD/LBD pro- teins. In Aim 1, we will determine the critical molecular residues in Hsp70 for blockage of ASyn oligomerization and toxicity. We will use state-of-the-art cryo-EM microscopic approaches to capture how ASyn binds to Hsp70 with atomic resolution and validate the role of this binding through mutational analysis in LBD-relevant biochem- ical and cellular ASyn oligomerization and toxicity assays. This will directly test our hypothesis that non-canonical Hsp70 engagement of ASyn underlies its activity in blocking ASyn oligomers and in providing protection against ASyn toxicity in neurons. In Aim 2, we will determine the impact of Hsp70 non-canonical action on oligomeriza- tion of additional AD and LBD-relevant protein species, including lipid-bound ASyn and tau and ...