Characterizing Microglial Response to Amyloid: From New Tools to New Molecules

Abstract

Microglia are a population of specialized,\ntissue-resident immune cells that make up around 10% of total cells in our\nbrain. They actively prune neuronal synapses, engulf cellular debris, and\nmisfolded protein aggregates such as the Alzheimer’s Disease (AD)-associated amyloid-beta\n(Aβ) by the process of phagocytosis. During AD, microglia are unable to\nphagocytose Aβ, perhaps due to the several disease-associated changes affecting\ntheir normal function. Functional molecules such as lipids and metabolites also\ninfluence microglial behavior but have primarily remained uncharacterized to\ndate. The overarching question of this work is, How do microglia become\ndysfunctional in chronic inflammation? To this end, we developed new\nchemical tools to better understand and investigate the microglial response to\nAβ in vitro and in vivo. Specifically, we introduce three new\ntools. (1) Recombinant human Aβ was developed via a rapid, refined, and robust\nmethod for expressing, purifying, and characterizing the protein. (2) A\npH-sensitive fluorophore conjugate of Aβ (called AβpH) was developed\nto identify and separate Aβ-specific phagocytic and non-phagocytic glial cells ex\nvivo and in vivo. (3) New lysosomal, mitochondrial, and nuclei-targeting\npH-activable fluorescent probes (called LysoShine, MitoShine, and NucShine,\nrespectively) to visualize subcellular organelles in live microglia. Next, we asked,\nWhat changes occur to the global lipid and metabolite profiles of microglia in\nthe presence of Aβ in vitro and in vivo? We screened 1500 lipids comprising\n10 lipid classes and 700 metabolites in microglia exposed to Aβ. We found significant\nchanges in specific lipid classes with acute and prolonged Aβ exposure. We also\nidentified a lipid-related protein that was differentially regulated due to Aβ in\nvivo. This new lipid reprogramming mechanism “turned on” in the presence of\ncellular stress was also present in microglia in the brains of the 5xFAD mouse\nmodel, suggesting a generic response to inflammation and toxicity. It is well\nknown that activated microglia induce reactive astrocytes during inflammation. Therefore,\nwe asked, What changes in proteins, lipids, and metabolites occur in astrocytes\ndue to their reactive state? We provide a comprehensive characterization of\nreactive astrocytes comprising 3660 proteins, 1500 lipids, and 700 metabolites.\nThese microglia and astrocytes datasets will be available to the scientific community\nas a web application. We propose a final model wherein the molecules secreted\nby reactive astrocytes may also induce lipid-related changes to the microglial\ncell state in inflammation. In conclusion, this thesis highlights chemical\nneuroimmunology as the new frontier of neuroscience propelled by the\ndevelopment of new chemical tools and techniques to characterize glial cell\nstates and function in neurodegeneration.