PROJECT SUMMARY/ABSTRACT Study rationale. Microglia, the resident innate immune cells of the central nervous system, are involved in the maintenance of tissue homeostasis and neuronal circuitry. These microglia specific processes are believed to become dysregulated during neurodegenerative diseases, such Alzheimer’s disease (AD). It has also been shown that many of the AD susceptibility genes are primarily expressed in microglia in the brain. Given these observations, it has been suggested that microglia would constitute an ideal point of intervention for future preventive and disease modifying therapies for AD. Nonetheless, the notion arose, that such a plastic and reactive cell type as the microglia would not exist in a single state in such a complex and diverse environment as the aging brain. This also implies the possibility, that the different subsets would be involved in different aspects of physiological and pathological processes. Indeed, in our recent single cell RNA sequencing study we have identified several distinct microglia subpopulations in the aged human brain, that showed (in an indirect analysis) divergent associations with clinical and histopathological characteristics of brain aging and AD. Intriguingly, the signature of one particular microglia subset, named Cluster 7, showed an inverse relationship with both tau pathology and clinical AD, suggesting a potential protective role for this microglia phenotype. Thus, the overarching goal of the proposed project is to explore the identity of Cluster 7 microglia and to understand how sex, aging, AD pathology and genetics influence its abundance in the human brain. Our hypothesis is that aging, AD pathology and genetic susceptibility converge on microglia and result in a differentiation deficit in particular towards the beneficial Cluster 7 phenotype, thus contributing to disease progression. We also hypothesize that this process is more pronounced in females than in males. Accordingly, the objective of the current proposal is: 1) to investigate how the abundance of this phenotype relates to histopathological and clinical features of brain aging and AD; 2) to understand what are the factors that might inhibit Cluster 7 microglial fate choice in AD; 3) to explore whether elevated Cluster 7 abundance is associated with cognitive resilience; and 3) to describe the phenotypic characteristics and epigenetic regulatory landscape of Cluster 7 microglia. These goals will be achieved through the following 3 aims: In Aim 1 we propose to measure in situ the abundance of Cluster 7 microglia subpopulation in a large number of aged individuals with and without AD dementia and will establish the relationship between the abundance of Cluster 7 and sex, age, histopathological features, AD endophenotypes, AD susceptibility alleles and measures of cognition. In Aim 2, we will explore the functional and phenotypic characteristics of ex vivo isolated Cluster 7 microglia. In Aim 3, we will investigate th...