Project Summary/Narrative This K08 Mentored Clinical Scientist Research Career Development Award application investigates the role of tau post-translational modifications on the cellular processes involved in the stereotyped spreading of tau pathology that occurs as Alzheimer’s disease progresses. The cellular mechanisms underlying this process include the cell-to-cell propagation of pathologic tau from a donor cell to a recipient cell, escape from the endosomal compartment in the recipient cell, and subsequent seeding of tau aggregation in the cytoplasm. Previous studies published in the literature, as well as preliminary data related to this project, suggest that tau post-translational modifications, especially phosphorylations and acetylations, influence these processes. The overall hypothesis of the project is that tau post-translational modifications found on pathologic tau in Alzheimer’s disease influence the cellular mechanisms involved in the spreading of tau pathology. The specific aims of the project focus on investigating the impact of disease-associated tau post-translational modifications on 1) the cell-to-cell propagation of tau, 2) the endosomal escape of tau, and 3) the seeding activity of tau. The research strategy for this project uses human induced pluripotent stem cell-derived neurons to study the processes outlined in the specific aims, and mutations mimicking post-translational modifications (pseudophosphorylations and pseudoacetylations) will be used to assess the contribution of defined combinations of specific residues. Specific Aim 1 uses an expression construct with the wildtype or mutant tau sequences to identify donor and recipient cells by flow cytometry to assess cell-to-cell propagation. Specific Aim 2 uses treatment of neurons with wildtype or mutant recombinant tau proteins and evaluation of endosomal membrane integrity and tau endosomal escape using immunocytochemistry with galectin-3 staining and a split luciferase assay, respectively. Specific Aim 3 uses tau Förster resonance energy transfer (FRET) biosensor cell models to detect tau seeding activity of the wildtype or mutant recombinant tau proteins or Alzheimer’s disease-derived tau preparations with or without in vitro dephosphorylation and/or deacetylation. The career development plan of this proposal is designed to prepare the applicant for independence as a scientist. The applicant will take courses that provide a conceptual framework for design, performance, and analysis of the experiments described above. Scientific communication skills will be further developed by coursework and experience in writing manuscripts/grants and presenting the results at scientific meetings. The applicant had assembled a team of mentors, both the primary mentor and Scientific Advisory Board, to assist in the applicant’s development over the course of the project. The combination of the research experience, career development activities, and mentorship the applicant recei...