Abstract Alzheimer's disease (AD) is the most common type of dementia affecting a large percentage of the elderly population. A hallmark event in AD is the brain accumulation of misfolded protein aggregates in the form of amyloid and tau pathology. Amyloid pathology comprises the predominantly extracellular accumulation of amyloid-β peptide (Aβ) in diverse type of amyloid deposits. Tau pathology appears as hyperphosphorylated Tau aggregates in the form of neurofibrillary tangles (NFT) in neuronal somata and neuropil threads in neuronal dendrites. Considerable evidence indicates that the accumulation of Aβ aggregates is the initiator of AD pathogenesis, but that Tau pathology is the main executor of neurodegeneration. Despite the substantial advances in understanding the genetic, cellular and molecular pathways implicated in AD, the disease is still incurable and have few (mostly symptomatic) options for treatment. In this project we propose the discovery and development of drugs for AD treatment by focusing on inhibiting the spreading of Tau aggregates. We plan to develop a highly relevant and robust screening assay to identify Tau inhibitors based on the protein misfolding cyclic amplification (PMCA) technology invented in our lab. PMCA reproduces the seeding/nucleation process that features the formation of all misfolded protein aggregates in the brain. We have recently developed a PMCA-based assay to study the seeded polymerization of Tau aggregates. Our goal is to adapt Tau-PMCA as a high-throughput primary screening test to identify drug candidates able to inhibit misfolding and aggregation of Tau. In the R61 phase we propose to develop a high- throughput version of Tau-PMCA (HT-Tau-PMCA) assay and validate it in terms of robustness, reproducibility and specificity using standard practices in the pharmaceutical industry. We will also perform a pilot screening of 2000 compounds to evaluate assay quality and estimate the rate of hit identification. In the R33 phase we propose to perform a large screening of a chemical library containing 100,000 compounds from diverse chemical classes. Hits identified by HT-Tau-PMCA will be characterized to obtain IC50 and to analyze specificity against other misfolded proteins. Hits with IC50 < 1 µM will be characterized by their drug-like properties using in silico approaches. The best hits will be used for studies using cellular and animal models of Tau propagation. This project has the potential to identify new chemical compounds with ability to inhibit Tau seeding and spreading that can be developed as a much needed therapy for AD.