The goal of this supplemental project is to extend the bioinformatic analyses and experimental validation of AMP-AD target networks, which are the focus of the parent grant. First (AIM1), we will generate single-cell longitudinal transcriptomic profiles of Drosophila AD models expressing human tau and secreted amyloid-β. These data will allow to distinguish gene expression profiles from distinct neuronal populations as well as different glia subtypes. These studies will also strongly complement ongoing AMP-AD single-cell gene expression profiles from human postmortem tissue. We will include rigorous experimental controls for tau / amyloid-β expression as well as longitudinal sampling to dissect out the specific contributions of age and AD pathologic species on cell-type specific gene expression changes in the brain. Initially we will include 2 time points (early and late) during disease progression. During the first year of the proposed supplement (Y4 of the parent R01 grant) we will generate Drosophila of the appropriate genotypes, extract mRNA, perform scRNAseq, and begin analyses of the results. During the second year of the supplement (Y5 of the parent R01 grant) we will complete analysis of the data, integrating with our findings from whole brain RNAseq and performing cross-species comparisons with AMP-AD scRNAseq. We will also perform independent experiments to confirm the most promising results from scRNAseq, such as immunofluorescence confocal microscopy, taking advantage of available antibodies, and reagents. Selected candidate causal drivers will also be manipulated using cell-type specific drivers, including glia and/or glutamatergic, GABAergic, cholinergic and dopaminergic neurons within Drosophila CNS to examine requirements for brain maintenance and/or function. Second (AIM2), we will extend experimental validation of computationally predicted AD causal genes from Drosophila to mammalian cells. All causal drivers identified in the tau Drosophila screen, will be tested in cultured mouse and human neural progenitor cells using shRNAs. Specifically, we will assess the impact of causal drivers on tau protein levels given the key role of tau accumulation AD pathogenesis. Our data shows that a subset of the identified tau modifier genes (e.g., Hippo pathway components, Nuak1) lower tau protein levels in Drosophila. Thus, we hypothesize that a subset of causal drivers identified in the ongoing AMP-AD screen modulate tau accumulation and this subset may be especially interesting from a therapeutic standpoint. During the first year of the proposed supplement (Y4 of the parent R01 grant) we will test the Hippo pathway genes in Neuro2A and human neural precursor cells plus all 44 tau modifier genes for their ability to modulate tau protein levels in the Drosophila brain using immunoblotting and Homogeneous Time Resolved Fluorescence approaches. During the second year of the supplement (Y5 of the parent R01 grant) we will test in Neuro2A and h...