Sex- and Diet- Dependent Microglial and Proteomic Responses to Chronic Diesel Exhaust Exposure in the Ldlr -/- Mouse Brain

Abstract

Background: Air pollution is one of the leading causes of mortality, contributing to about 6.7 million premature deaths annually. Diesel exhaust (DE) is a significant contributor to particulate matter (PM) in urban traffic-related air pollution, resulting in adverse respiratory, cardiovascular, and reproductive health effects. Research has suggested that PM can also affect the brain and central nervous system, triggering neurodegeneration. Microglia, the brain's resident immune cells, play a crucial role in regulating PM-induced toxicity. To protect the brain from damage, resting microglia are activated to eliminate foreign substances and dead cells, while initiating an inflammatory response. Though initially beneficial to maintain homeostasis and repair damage, prolonged exposure to PM can cause microglia to promote a pro-inflammatory environment due to oxidative stress and cell death, leading to disease escalation. Vulnerable and susceptible populations have an increased risk of PM-induced toxicity. People with genetic mutations, like familial hypercholesterolemia (FH), are at an increased risk of neurodegenerative disease due to their increased risk of cardiovascular disease. While there is increasing research to uncover how PM impacts neurodegeneration, the specific molecular mechanisms are not well understood. The objective of this project was to characterize the effects of chronic DE exposure and a high-fat diet on neuroinflammatory and proteomic responses in the hippocampus and olfactory bulb of a mouse model of FH. Based on publications and our laboratory's preliminary data, we hypothesize that chronic DE exposure will induce sex-specific changes in microglial states and protein pathways, particularly in the hippocampus and olfactory bulb, and that these changes will be exacerbated by HFD in hypercholesterolemic mice.Methods: This study examined mechanisms of PM-induced neuroinflammation and cognitive dysfunction, focusing on proteomics and microglia. Six-week-old low-density lipoprotein receptor knockout (Ldlr-/-) mice, a model of FH, were exposed to freshly generated DE (~250 μg/m3) or filtered air (FA) over 18 weeks. Mice (n=11-13 per sex and exposure) were fed a high-fat (HFD) or chow diet and exposed for 6 h/day, 5 days/week. At the end of 18 weeks, mice were sacrificed and perfused with PBS. Left brain hemispheres were fixed, cryopreserved, and used for immunofluorescence. Confocal images (40x) were taken for each group (n = 2) and an average cell count was taken. From each 40x image, a 3x zoom image was taken, and soma and arborization area were measured for 3 random microglia. The hippocampus and olfactory bulb were isolated from right brain hemispheres, flash frozen in liquid nitrogen, analyzed for proteomics via data-independent acquisition liquid chromatography-tandem mass spectrometry (DIA LC-MS/MS). Resulting datasets were analyzed using the Advaita iPathwayGuide software. Results: An overall decrease in microglia was found in the DE groups when compared to the FA groups, highlighting possible microglial dysfunction based on diet and DE exposure. The HFD DE female (DEF) group had the largest decrease (p= 0.0463) in microglial cell count (42%) and the largest increase in soma (~19%) and arborization area (~66%). Protein analysis revealed significant changes (FDR< 0.05) based on exposure (FA or DE) in the olfactory bulb between the HFD DE and HFD FA female groups. In the hippocampus and olfactory bulb of male mice, protein changes observed were based on diet when comparing HFD DE and control chow DE groups