Project Summary Epidemiological studies have reported a strong positive association between traffic-related air pollution (TRAP) and risk of neurodegenerative disease, including Alzheimer’s Disease (AD) and AD-related dementias (ADRD). Since human exposure to TRAP will continue to increase as a result of expanding global industrialization and urbanization, there is an urgent need to identify which components in TRAP endanger the human brain, and to identify how TRAP promotes neurodegenerative disease. The work described in this Diversity supplement will address these data gaps by leveraging a novel in vitro neuron-astrocyte-microglia triculture model that more faithfully recapitulates in vivo neuroinflammatory responses than standard neuron monocultures or neuron- astrocyte co-cultures to test the hypothesis that TRAP-derived fine particulate matter (PM2.5) interacts directly with microglia in the brain to promote AD-relevant pathology. The scientific premise for this hypothesis includes experimental evidence previously reported by our research team demonstrating that chronic exposure to ambient TRAP collected from a heavily-trafficked tunnel in Northern California and delivered unchanged in real-time to rats in an adjacent vivarium: (1) decreased the time-to onset and increased the magnitude of AD/ADRD-relevant phenotypes, including neuron cell loss in the entorhinal cortex and hippocampus; (2) TRAP promoted microglial activation, but had no effect on astrogliosis as determined by quantitative immunohistochemical analyses of IBA- 1/CD68 and GFAP expression; and (3) significantly increased the number of particles in the ultrafine PM size range present in the hippocampus of TRAP-exposed animals relative to filtered air controls. The research goals of this Diversity supplement are to (1) compare the neurotoxicity of TRAP-derived PM2.5 vs. TRAP-derived soluble gases in the triculture model; (2) determine whether microglia mediate the neurotoxic effects of TRAP- derived PM; and (3) assess the influence of sex and genotype on microglia response to TRAP. This research is complementary to and extends the research described in the parent grant, which is focused on examining the influence of chronic exposure to different sources of TRAP vehicular emissions on AD-relevant pathology in an animal model and examining the role of the lung-brain axis in mediating the effects of TRAP on the brain. The training goals of this Diversity supplement include: (1) develop the trainee’s knowledge and technical skill set to enable them to successfully conduct research on environmental influences on neurodegenerative disease; (2) guide the trainee’s research activity to ensure the generation of data needed to support their preparation of a competitive F31 application and advance to candidacy; (3) enhance the trainee’s professional skills; and (4) actively work with the trainee to build their professional networks to enhance their likelihood of transitioning to an independ...