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Microglial Lipidome and Metabolome with Amyloid Beta Exposure

ABSTRACT: Microglia are specialized immune cells in the brain that eliminate misfolded protein deposits and cellular debris by phagocytosis. In Alzheimers disease, microglia clear the amyloid beta aggregates during disease onset but are unable to phagocytose them from the tissue environment during the late stages of the disease contributing to chronic inflammation. Lipids and metabolites orchestrate critical regulation events within the cell or by their secretion into the cellular microenvironment, yet, the effect of abeta on microglial lipidome and metabolome has not been well characterized till date. In this study, we used Multiple-Reaction Monitoring (MRM)-profiling to evaluate, for the first time, the lipid and metabolite changes in abeta-activated microglia that were isolated from mouse brains over 24 hours. We also developed a new bioinformatics pipeline to identify the statistically significant lipids and metabolites in abeta-treated microglial cells and condition medium that were differentially regulated compared to the vehicle controls. Triacylglycerides and phosphatidylglycerols were the most abundant lipid classes, whereas long-chain free fatty acids with 26 to 30 acyl chain carbon atoms were consistently decreased with abeta-treatment over time. Further, 42 significant metabolites were identified in cells. L-2-aminobutyric acid, hydroxybutyric acid, inosine, alanine, etc. were differentially regulated with abeta treatment suggesting changes in alanine, aspartate and glutamate metabolism, and arginine biosynthesis pathways. Selected metabolites were identified in the microglial conditioned medium, including stearic acid, tryptophan, palmitic acid, etc. that have been shown to affect the phagocytic properties of peripheral macrophages and may also play a role in the microglial phagocytic response. Thus, characterizing the lipidome and metabolome of microglia due to abeta identifies critical molecules involved in their immune response and highlight the molecular mechanisms of phagocytic function as well as dysfunction associated with AD.

INSTRUMENT(S): 6545 Q-TOF LC/MS, 6410 Triple Quadrupole LC/MS

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Gaurav Chopra

PROVIDER: MSV000089458 | MassIVE | Wed May 11 09:37:00 BST 2022

REPOSITORIES: MassIVE

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Project description:Microglia are specialized immune cells in the brain that eliminate misfolded protein deposits and cellular debris by phagocytosis. In Alzheimers disease, microglia clear the amyloid beta aggregates during disease onset but are unable to phagocytose them from the tissue environment during the late stages of the disease contributing to chronic inflammation. Lipids and metabolites orchestrate critical regulation events within the cell or by their secretion into the cellular microenvironment, yet, the effect of abeta on microglial lipidome and metabolome has not been well characterized till date. In this study, we used Multiple-Reaction Monitoring (MRM)-profiling to evaluate, for the first time, the lipid and metabolite changes in abeta-activated microglia that were isolated from mouse brains over 24 hours. We also developed a new bioinformatics pipeline to identify the statistically significant lipids and metabolites in abeta-treated microglial cells and condition medium that were differentially regulated compared to the vehicle controls. Triacylglycerides and phosphatidylglycerols were the most abundant lipid classes, whereas long-chain free fatty acids with 26 to 30 acyl chain carbon atoms were consistently decreased with abeta-treatment over time. Further, 42 significant metabolites were identified in cells. L-2-aminobutyric acid, hydroxybutyric acid, inosine, alanine, etc. were differentially regulated with abeta treatment suggesting changes in alanine, aspartate and glutamate metabolism, and arginine biosynthesis pathways. Selected metabolites were identified in the microglial conditioned medium, including stearic acid, tryptophan, palmitic acid, etc. that have been shown to affect the phagocytic properties of peripheral macrophages and may also play a role in the microglial phagocytic response. Thus, characterizing the lipidome and metabolome of microglia due to abeta identifies critical molecules involved in their immune response and highlight the molecular mechanisms of phagocytic function as well as dysfunction associated with AD.

Project description:Microglia are innate immune cells of the brain that perform phagocytic and inflammatory functions in disease conditions. Transcriptomic studies of acutely-isolated microglia have provided novel insights into their molecular and functional diversity in homeostatic and neurodegenerative disease states. State-of-the-art mass spectrometric methods can comprehensively characterize proteomic alterations in microglia in neurodegenerative disorders, potentially providing novel functionally-relevant molecular insights that are not provided by transcriptomics. However, proteomic profiling of adult primary microglia in neurodegenerative disease conditions has not been performed. We performed quantitative proteomic analyses of purified CD11b+ acutely-isolated microglia adult mice in normal, acute neuroinflammatory (LPS-treatment) and chronic neurodegenerative states (5xFAD model of Alzheimer’s disease [AD]) using tandem mass tag mass spectrometry. Differential expression analyses were performed to characterize specific microglial proteomic changes in 5xFAD mice and identify overlap with LPS-induced pro-inflammatory changes. Our results were also contrasted with existing proteomic data from wild-type mouse microglia and from existing microglial transcriptomic data from wild-type and 5xFAD mice. Neuropathological validation studies of select proteins were performed in human AD and 5xFAD brains. Of 4,133 proteins identified, 187 microglial proteins were differentially expressed in the 5xFAD mouse model of AD pathology, including proteins with previously known (Apoe, Clu and Htra1) as well as previously unreported relevance to AD biology (Cotl1 and Hexb). Proteins upregulated in 5xFAD microglia shared significant overlap with pro-inflammatory changes observed in LPS-treated mice. Several proteins increased in human AD brain were also upregulated by 5xFAD microglia (Aβ peptide, Apoe, Htra1, Cotl1 and Clu). Cotl1 was identified as a novel microglia-specific marker with increased expression and strong association with AD neuropathology. Apoe protein was also detected within plaque-associated microglia in which Apoe and Aβ were highly co-localized suggesting a role for Apoe in phagocytic clearance of Aβ. We report the first comprehensive comparative proteomic study of adult mouse microglia derived from acute neuroinflammatory and AD models, representing a valuable resource to the neuroscience research community. We highlight shared and unique microglial proteomic changes in acute neuroinflammatory, aging and AD mouse models in addition to identifying novel roles for microglial proteins in human neurodegeneration.

Project description:Microglia, the resident immune cells of the central nervous system (CNS), have two distinct phenotypes in the developing brain: amoeboid form, known to be amoeboid microglial cells (AMC) and ramified form, known to be ramified microglial cells (RMC) alongside several intermediate forms. The AMC are characterized by being proliferative, phagocytic and migratory whereas the RMC are quiescent and exhibit a slow turnover rate. The AMC transform into RMC with advancing age, and this transformation is indicative of the gradual shift in the microglial functions. Both AMC and RMC respond to CNS inflammation, and they become hypertrophic when they are activated by trauma, infection or neurodegenerative stimuli. The molecular mechanisms and functional significance of morphological transformation of microglia during normal development and in disease conditions is not clear. It is hypothesized that AMC and RMC are functionally regulated by a specific set of genes encoding various signaling molecules and transcription factors. To address this, we carried out cDNA microarray analysis using lectin-labeled AMC and RMC isolated from frozen tissue sections of the corpus callosum of 5-day and 4-week old rat brain respectively, by laser capture microdissection (LCM). The global gene expression profiles of both microglial phenotypes were compared and the differentially expressed genes in AMC and RMC were clustered based on their functional annotations. This genome wide comparative analysis helps in identifying genes that are specific to AMC and RMC. The novel and specific molecules identified in both microglial phenotypes can be targeted for therapeutic purposes in developing and adult brain diseases. We used microarrays to identify the genes specific to amoeboid and ramified microglia. RNA was isolated from the laser-captured amoeboid and ramified microglia from the corpus callosum of 5-day and 4-week old rat brain. The RNA was hybridised onto Affymetrix Rat 230 2.0 array.

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Microglial Lipidome and Metabolome with Amyloid Beta Exposure

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