PROJECT SUMMARY/ABSTRACT This supplement complements our parent grant: NIDCD R21DC021275-01 “Investigating the origin and functional properties of immune cells in noise-induced hearing loss”, and extends the original studies to include the 5XFAD mouse model of Alzheimer's disease (AD). Hearing loss has been identified as one of eight risk factors for AD by the Center for Disease Control and Prevention and accounts for 8% of AD cases worldwide. However, the pathological link between hearing loss and AD has not been established. It is not known whether hearing loss leads to AD, or if hearing loss and AD share a common underlying mechanism. Since individuals with hearing impairment have a 30-40% rate of accelerated cognitive decline and an increased risk of AD compared to individuals with normal hearing, and noise damage is one of the leading causes of hearing loss, it is crucial to understand how/if these conditions share a common pathology. The parent project, started in July 2023, is investigating the cellular identities and functional properties of macrophages after noise damage in young and aged mice. In the parent project, we are testing the hypothesis that macrophages from different origins are involved in noise-induced hearing loss depending on the age of the animal and stage of the disease (acute, recovery or recovered). During the course of the study, we made the observation that cochlea contain several tissue-resident macrophage populations. We discovered that these cochlea tissue-resident macrophage populations express genes responsible for “synaptic pruning” and genes such as TREM2 that have been identified as AD susceptibility genes. Interestingly, microglia, the brain immune cells, also express these genes. Research in the past decade has the loss discovered shown that microglia are critically involved in pathogenesis of AD and responsible for several aspects of AD athology such as early mediators of synapse and dysfunction. Whether the new cochlea tissue-resident macrophage ubpopulations that we have and microglia represent the pathological link between hearing loss and AD is unknown. In p s the current supplement, we propose to perform single-cell RNAseq analysis of cochlea macrophages and brain microglia (isolated by cell sorting from 5XFAD mice) at 6-weeks of age prior to the formation of AD pathology (amyloid plaques) and hearing loss and at four months when there is severe amyloid pathology and hearing loss. Single- cell RNAseq will provide new information on cochlea macrophage heterogeneity in AD before and after the development of pathology. Furthermore, differential gene expression analysis of cochlea macrophages and brain microglia will help to test the hypothesis whether cochlea tissue-resident macrophages and microglia are associated with a common pathological pathway that links hearing loss and AD. Specific Aim 1. Characterize myeloid cell heterogeneity, and define phenotype and spatial distribution of tissue- resident...