Project Summary. Glycogen synthase kinase 3 (GSK3) is considered a key player in the pathophysiology of Alzheimer’s disease (AD) since dysregulation of this enzyme influences all the major AD hallmarks, including tau phosphorylation, amyloid-β production, neurogenesis, inflammation and synaptic function. Therefore pharmacological modulation of GSK3 represents an attractive therapeutic approach for the treatment of AD. Positron emission tomography (PET) is capable of quantifying biochemical processes in vivo, and a suitable GSK3 ligand would substantially improve our understanding of GSK3-mediated signaling pathway under different pathophysiological AD conditions, otherwise inaccessible by ex vivo (destructive) analysis. Quantification of GSK3 in living brain by PET would provide the assessment of distribution, target engagement and dose occupancy of new GSK3-targeted neurotherapeutics. To date, no successful examples have been demonstrated to image GSK3 in human for drug discovery and clinical use, representing a significant deficiency of our ability to study this target in vivo. Therefore, we propose to develop a novel PET ligand that can fill this void, as the first translational imaging tool. We are the first groups to develop GSK3-specific ligands in cross-species PET studies, including the first selective ligand [11C]PF-367. However, this ligand was discontinued due to low-to-moderate brain uptake and marginal binding specificity in vivo. In our 2nd generation, we identified a lead molecule, GSK3-817, which showed high binding affinity and excellent selectivity. An 18F-isotopologue of GSK3-817 was synthesized and preliminary PET imaging studies confirmed that we have overcome two major obstacles for GSK3-specific kinase ligand development by achieving: 1) substantially-improved brain penetration (≥1 SUV brain uptake) and 2) reasonable target specificity. Though GSK3- 817 is a promising lead molecule for the development of new GSK3-targeted PET ligands, further optimization for improved binding specificity and proper brain kinetics are sought for translational cross-species (rodents and nonhuman primates) imaging studies to achieve optimal GSK3 quantification for drug discovery and clinical translation for AD patients. On the basis that GSK3-817 serves a validated lead for medicinal chemistry optimization, as specific goals, we will design and prepare a focused library of GSK3-specific modulators amenable for labeling with 11C or 18F (preferred), and evaluate their ability to quantify GSK3 expression and changes during drug challenge in rodents and nonhuman primates, as well as autoradiography and biological validation in postmortem human brain tissues. The impact of this work is not only to develop the first successful highly-specific GSK3 PET ligand for the study of neurodegenerative disease-related biological processes, but also ultimately, via PET imaging validation in higher species, to advance this ligand for potential clinical transl...