Abstract Protein aggregation is a hallmark of diverse neurodegenerative diseases, including Alzheimer, Huntington and Parkinson Diseases (AD, HD and PD) which will be studied in this Program Project, but little is known about the role of aggregates in neurotoxicity or clinical manifestations. Different brain regions are affected in each disease, underlying their distinctive pathologies, and different diagnostic proteins are detected immunologically for each (presumably reflecting distinct proteins that initiate or feed aggregates due to their high abundance or post-synthetic modifications in the vulnerable tissue). Increasingly, however, proteins “characteristic” of one neuropathy have been detected in other disease aggregates, leading us to the hypothesis that a common aggregation mechanism is involved. To learn what features cause proteins to coalesce with the “seed proteins” (i. e., the mutated and/or misfolded protein that initiates aggregation), we propose a systematic proteomic comparison of aggregates purified from affected tissue of each disease, using unaffected tissue from patients and corresponding areas of normal brain as controls. Our preliminary proteomic analyses show that immuno- affinity for tau or Aβ allows isolation of two distinctive types of aggregates, which nevertheless share many proteins in common. Aggregates derived from AD vs. age-matched-control (AMC) hippocampus differ strikingly in protein composition, whether they bind tau or Aβ antibodies. These results are supported by images showing co-localization of proteins by immunohistochemical and proximity-ligation methods, and by C. elegans models of AD in which orthologs of identified AD-aggregate proteins contribute functionally to aggregation and associated traits. In Aim 1, proteomic analyses of proteins (and their most common modifications) from AD, PD, HD and AMC tissues will enable us to seek features common to two or three diseases or unique to each. We will also seek “leading indicators” of AD, in individuals at highest risk (based on ApoE4 alleles, age or Down Syndrome) and in 3xTg-AD mice prior to overt AD pathology. We will then analyze aggregates from various cell and worm models, to prioritize these based on their similarity to the disease aggregates they mimic. In Aim 2, we will identify cross-linked peptides from aggregates, defining protein:protein contacts that Project 1 will attempt to confirm by immuno-stain colocalization, and by proximity-ligation PCR. These results will guide molecular-dynamic modeling of aggregate formation, in which we can assess the energies (‒ΔE and ‒ΔG) driving and stabilizing protein accretion, and test the effects of mutating putative contact residues. Key aggregate proteins seen in multiple diseases (Aim 1) will be tested for functional roles and pathways employed, using RNAi in neuroblastoma cells (SY5Y-APP) and worm models. NSAID-derived compounds from Core D will be screened in Aim 3 for protection of SY5Y-APP cells from ...