PROJECT 2: PROJECT SUMMARY/ABSTRACT Alzheimer’s Disease (AD) is the most prevalent neurodegenerative disease of aging, and results in a variety of symptoms, the most prominent of which is progressive cognitive decline. Whereas the primary pathological proteinopathies associated with AD are aggregates of amyloid-beta (plaques) and tau (neurofibrillary tangles), the appearance, density, and distribution of these proteinopathies shows substantial heterogeneity across individuals and brain regions. In addition, individuals often display co-occurrence of these two proteinopathies with other protein aggregates such as TDP43 and alpha-synuclein; these latter two are primarily associated with diseases such as Amyotrophic Lateral Sclerosis, Frontotemporal Dementia and Parkinson’s Disease, but their occurrence in AD is not rare. From our bulk and single-nucleus RNA-seq studies on post-mortem tissue, we have identified specific cellular signatures/putative subtypes whose abundance is associated with tangles and plaques. However, these bulk and single-nucleus experimental approaches do not preserve spatial information about how these cell types are localized (or not) around pathological inclusions, and thus generate an incomplete picture of how the cellular structure of the aged brain is altered in the presence of AD pathology. Here, we aim to examine the spatial distribution and organization of these cell types directly in intact human brain tissue using highly-multiplexed spatial methods. We propose a combination of novel but demonstrably scalable protein and gene expression approaches to study large volumes (300-micron thickness) of human tissue with combinations of these proteinopathies. These large volumes are necessary to study the full impact of protein aggregates on the “highly local” environment. Specifically, we use a combination of highly multiplexed antibody staining and genome-wide spatial transcriptomics methods to examine our hypothesis on the reorganization of neuronal and glial cell types around pathological inclusions, with a focus on the two major AD-associated proteinopathies (amyloid and tau). Ultimately, this will generate a high-resolution atlas of cell types and gene expression signatures in aged human brain tissue that are altered in the presence of protein aggregates commonly found in AD, complementing many of the existing and ongoing bulk and single-nucleus RNA-seq studies in the field of human neurodegeneration research.