Project Summary Alzheimer’s Disease (AD) presents a striking example of the selective cell tropism common to most neurodegenerative diseases. Early clinical symptoms (such as memory loss) are caused by the selective buildup of tau pathology and by the degeneration of principal neurons of the entorhinal cortex layer II (ECII). Understanding pathological lesion formation in ECII might be key to design disease-modifying therapeutics, since pathological tau seeds spread from ECII to other brain regions in later stages of AD. Additionally, investigating the most AD-vulnerable neurons of the brain could reveal crucial steps in the molecular events that lead to degeneration. In previous work, we molecularly profiled ECII neurons and compared them to neurons more resistant to the disease using a systems-level framework. We identified novel genes and pathways predicted to drive neuronal pathology. But we are still missing an in-depth understanding of both the particular proteoforms of tau accumulating in the EC, and the concomitant gene expression changes occurring in ECII neurons and surrounding glial and neuronal cells. We propose here to study postmortem TEC (transentorhinal cortex – the subarea of the EC where the very first neurofibrillary tangles appear), EC, and prefrontal cortex (PFC) from asymptomatic individuals with either no amyloid and tau pathology (Braak stage 0), or with amyloid and early stage neurofibrillary tangle pathology (Braak stage I/II). With these samples, we will perform single-nucleus combinatorial indexing RNA-sequencing, in order to profile all cell types and subtypes. Cross-referencing the single-nucleus RNA-sequencing dataset with a fully annotated mouse single- nucleus RNA-sequencing dataset of EC will allow to identify nuclei from vulnerable ECII neurons. Performing cell type abundance and differential expression analysis in three regions with increasing vulnerability to the disease (TEC – EC – PFC) will yield a picture of the sequential changes occurring during early disease. Samples will also be subjected to targeted mass spectrometry analysis to detect Aβ peptides and a number of tau phosphorylation sites, cleavage and splice isoforms to identify potential pathological proteoforms specific to early AD. Jointly analyzing the proteomics and transcriptomics dataset will allow us to find specific genes correlated with the most salient proteoforms. This multimodal catalog will also be analyzed jointly with our previously described ECII functional network, and altogether should yield exciting insight into the earliest events occurring in vulnerable neurons as AD-type neuropathology unfolds, uncovering genes both dysregulated and predicted to drive downstream tau pathology.