THE SYNAPTIC TYPE LANDSCAPE OF ALZHEIMER'S DISEASE VISUALIZED BY MULTIPLEX EXPANSION SYNAPTIC NANOSCOPY Abstract Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder, with high morbidity and mortality and high costs toward patient care and management, posing a major global public health issue. In addition, the link between AD and neuroinflammation suggests that this burden may significantly increase in the years following the COVID-19 pandemic. AD is characterized by memory loss, cognitive decline, and devastating neurodegeneration associated with synaptic dysfunction and loss. Genome-Wide Association Studies implicate diverse molecular pathways for human AD disease, but the way that these genetic results relate to synaptic pathology is unclear. In situ proteomic profiling of synapses across the brain is critical to shed light on potential mechanisms and therapies. Unfortunately, conventional bulk and single-cell biochemical and sequencing assays lack spatial resolution for fine-scale synaptic analysis as well as the isolation of discrete synaptic types that may be differentially affected in AD. Although super-resolution fluorescence imaging methods can offer sufficient spatial resolution, multicolor imaging is limited to ~4 protein targets due to spectral overlap, which cannot reveal the convoluted pictures of synaptic pathology in AD. Built upon our innovative molecular imaging platform supported by the parent DP2, here we propose a nanoscale synapse imaging method that will determine high- dimensional protein location and protein-protein interactions at synapse in both normal and pathological states with commercially available reagents, for both clinical AD samples as well as in mouse models of amyloidogenesis. The outcome of our work will lead to a broadly useful in situ proteomic tool for quantification of neuronal synapse types available to diverse neuropathology laboratories and will foster a better understanding of the complexity of molecular mechanisms of AD for development of novel therapeutics.