Project Summary Abstract The spatial and molecular details of cell type vulnerability during Alzheimer’s disease (AD) progression remain unclear. The objective of this project is to apply spatial profiling methods to brain tissue from donors at multiple stages of Alzheimer’s disease to describe changes in cell type composition, location, and gene expression as pathology advances across brain regions. The field of spatial transcriptomics is highly dynamic and rapidly evolving, with rapid progress and now commercialization that offers the potential to better achieve the original aims of the project. Multiplexed Error Robust In Situ Hybridization (MERFISH) is a technique capable of profiling hundreds or thousands of genes in tissue sections, which enables identification of transcriptomically- defined cell types and provides a quantitative assessment of gene expression without disrupting tissue structure. Recently a commercially developed version of MERFISH has become available in the form of MERSCOPE by Vizgen, which provides the benefits of an all-in-one system capable of reliable high throughput spatial profiling of large tissue sections. The MERSCOPE system represents a significant advance in technology that was not available at the time of the grant’s funding and can dramatically accelerate the achievement of our aims to profile changes in tissue microarchitecture associated with AD. Pilot data from this platform derived from both neurotypical and AD-affected donor brain tissue has shown that this approach is an effective and comprehensive method for profiling cell type composition and gene expression. Importantly, the Vizgen MERSCOPE platform is compatible with imaging both gene and protein expression. By combining immunohistochemistry (IHC) and MERFISH with this system, we will be able to correlate sites of pathological protein aggregation with proximity to specific cell types at distinct stages of disease. We have developed IHC chemistry compatible with MERFISH profiling and can combine these methods for use on the MERSCOPE. The MERSCOPE platform will allow the project to deliver greater scientific impact by significantly accelerating the original Project 3 grant aims. It will enable us to profile tissue from multiple disease stages rapidly, while placing cell type and gene expression in the context of disease progression and pathological degeneration of tissue.