PROJECT 1: PROJECT SUMMARY/ABSTRACT Aging is characterized by the accumulation of molecular and cellular damage resulting in decreasing cellular functionality; diseases that are particularly associated with aging include cerebrovascular disease and neurodegenerative diseases. Although focus has been on individual proteinopathies and cerebrovascular defects that occur more frequently with age, little is known about the local effects of aging on gene expression signatures and how these effects impact cellular composition and increase susceptibility to disease. Here, we focus on how ageing alters the cellular environment in three distinct regions of the brain, in patients with minimal or no pathology, that are known to be susceptible to the effects of protein and/or vascular pathologies in later life: superior temporal gyrus, dorsolateral prefrontal cortex, and calcarine cortex. We hypothesize that normal aging will have local and global effects on cell type composition and associated gene expression/molecular signatures, and that understanding these attributes will allow us to form a better understanding of why divergence from this state may result in pathology in these vulnerable regions. The aim of this project is to generate a spatial atlas of normal aging using individuals spanning an age range from 20- 90, all with minimal to no underlying pathology. To achieve this goal, we will use immunohistochemical (4i) and unbiased transcriptomics (10x Visium ST) techniques to generate a large-volume (300-micron thickness) 3-D atlas of three key brain regions vulnerable to pathology in later life from 30 individuals. We aim to derive cell type- and molecular signature-specific maps for each subject, integrating these data into a computational framework to characterize changes in local and global signatures with respect to age. We will then validate high-value associations derived from this framework in a replication cohort of 20 individuals. Using this approach, we hope to provide an unprecedented set of cell type and gene expression signature maps that represent the spectrum of normal aging.