PROJECT II: Summary/Abstract Pathological alpha-Synuclein (aSyn) accumulates in neurons as Lewy bodies (LBs) and Lewy neurites (LNs) in Lewy body diseases (LBD) including dementia with Lewy bodies (DLB), Parkinson’s disease (PD), PD with dementia (PDD), and in oligodendrocytes as glial cytoplasmic inclusions (GCIs) in multiple system atrophy (MSA). Cognitive deficit is common in LBD and >50% of cases have Aβ plaques and neurofibrillary tangles (NFTs) but how Alzheimer’s disease (AD) co-pathology contributes to LBD is not entirely clear. This diversity of aSyn pathology in brains of neurodegenerative disease patients supports the strain hypothesis of synucleinopathies wherein pathological aSyn adopts different conformations or strains that drive clinical and pathological heterogeneity of these disorders. aSyn pre-formed fibrils (PFFs) has been widely used to study these neurodegenerative diseases, but recently, we revealed that they do not fully recapitulate the biological behavior of LBs [1]. Significantly, we have purified and successfully amplified LBs from human brain, demonstrating that the pathological phenotypes observed in cultured neurons is maintained during the amplification process. Thus, our application will provide a more in-depth characterization of aSyn ultrastructure from different pathologies and decipher how these entities are internalized and hypothetically deregulate lysosome function. We will use a combination of structural, biochemical, and cell biological approaches to determine the molecular signatures of aSyn strains and elucidate the mechanisms of amplified LBs designated as LBs-P1 and PFFs and to explain why they have different seeding abilities. By identifying aSyn signatures, we will be able to confidently determine the molecular mechanism necessary for their internalization. Project II’s studies will provide unprecedented insights into the molecular architecture and biological activity of aSyn derived from human brain tissue of DLB, PD and AD patients, allowing us to better understand the heterogeneous nature of the synucleinopathies.