Project Summary Conformational variations in α-syn fibrils are proposed to be implicated in the emergence of distinct diseases within synucleinopathies, including Lewy body dementia (LBD), Parkinsons’s disease (PD), and multiple system atrophy (MSA), suggesting a molecular fingerprint closely connected to clinical diagnosis. However, the mechanisms underlying the appearance of these disease-specific conformations and their impact on the progression of distinct pathologies in synucleinopathies remain largely unclear. Motivated by the existing knowledge gap between structural polymorphism and disease entities, the goal of this proposed research is to investigate how α-syn pathology propagates in a disease-specific manner, by focusing on the molecular interactions between α-syn fibril structures and diverse cellular environments. To achieve this, we will employ a multidisciplinary approach based on the combination of biophysical approach, molecular biology, and human organ-on-a-chip technology. Building upon our complementary expertise in neurodegenerative amyloid fibril research and human organ-chip development, we will produce various forms of α-syn fibril seeds, characterize their conformations, and investigate their functions in human vascularized brain-on-chips with an unprecedented physiological realism. The central hypothesis posits that the membrane-mediated polymorphisms of α-syn fibrils are a key factor in the emergence of the distinctive pathological features of synucleinopathies, including LBD and PD, through the molecular interactions between α-syn fibrils and cellular membranes. These interactions are significantly influenced by the distinct conformations of α-syn fibrils and the varying membrane conditions found in different types of brain cells. This hypothesis will be tested by pursuing three specific aims: 1) Generate the polymorphic α-syn fibrils in the presence of various physiologically relevant membranes and characterize their functional properties; 2) Create microengineered 3D culture arrays for on-chip production of vascularized human mid-brain tissues and validate the propagation of α-syn pathology; and 3) Explore the cell-type specific pathological impacts of lipid-associated polymorphic α-syn fibrils and compare the molecular structures and interactions of these polymorphic α-syn fibrils. The expected outcomes will provide novel insights into the molecular mechanisms underlying conformation-dependent neuropathology. Consequently, these findings will contribute to the design and development of small molecules aimed at either inhibiting aggregations or detecting neurotoxic aggregates. Additionally, the brain-on-a-chip established in this study has the potential to serve as a promising drug screening platform capable of faithfully replicating cellular responses to pharmaceutical agents. Ultimately, this study will aid in the development of advanced diagnostic and therapeutic approaches aimed at synucleinopathies, thereby miti...