The basis for the onset and progression of Parkinson's disease (PD) and related dementias remains unknown, but the formation and spread of aggregates of the protein alpha-synuclein (aSyn) between neurons has been identified as a likely mechanism. Thus, understanding the molecular underpinnings of this prion-like spread is a key step that would set the stage for developing therapies to delay or alleviate PD-related motor dysfunction and dementia. However, to date, there has been no viable method to comprehensively investigate the under- lying phenomena of aggregation in live cells, and certainly not in vivo. The long-term goal of this research is to define the molecular mechanisms that contribute to neurodegeneration in people with PD and related de- mentias, in order to stimulate the development of new therapies. The overall objective of this application is to establish a role for aSyn-mediated membrane permeabilization in the spread of aSyn neuropathology in PD. The central hypothesis is that aSyn oligomers derived from internalized preformed fibrils (PFFs) facilitate the endocytic escape of aSyn seeds by permeabilizing the endocytic membrane from within. This hypothesis will be addressed with the following specific aims: (i) Define aSyn assembly states in different subcellular loca- tions of PFF-treated neurons; and (ii) Define aSyn assembly states at various stages of PFF-mediated aSyn propagation in vivo. The project entails fluorescence lifetime imaging microscopy (FLIM) studies with neurons, where lifetime depends on aggregation level, as well as steps to application in vivo, enabled by high-resolution three-dimensional fluorescence localization with point-wise lifetime information (and hence insight into the aSyn self-assembly state) at various positions in the brain over time. Notably, fluorescence quenching and hence lifetime reduction, similar to what occurs in fluorescence resonance energy transfer (FRET), has been shown by the group to occur when aSyn fused to a fluorescent protein (aSyn-FP) undergoes self-assembly to a beta- sheet-rich, aggregated state, with a lifetime that reduces with increasing aggregate size. This provides a means to study aSyn aggregate formation and spread using high-resolution FLIM (Aim 1). Drawing on the group's conceptualization and demonstration of a means to image fluorescence parameters in vivo and through heavy scatter, and also at high resolution using computational imaging with localization, fluorescence lifetime param- eters determined at a set of locations in the brain should yield aSyn aggregate and spread information (Aim 2). Upon completion of this project, live-cell studies will have allowed aSyn aggregation and spread to be character- ized, which should lead to understanding of the molecular process, and critical steps to optical sensing of aSyn aggregate spread in the whole brain of animals will have been achieved. This approach is innovative because it is focused on new technologies and research avenues r...