Dataset Information

Proteomic analysis of alcohol and LPS-treated primary mouse microglia

ABSTRACT: Microglia, the resident immune cells of the brain, can exhibit a broad range of activation phenotypes, many of which have been implicated in several diseases and disorders of the central nervous system including alcohol use disorders and disorders. By utilizing a method optimized for sensitive and rapid quantitative proteomic analysis of microglia involving suspension trapping (S-Trap), we were able to produce efficient and reproducible protein extraction from low cell yielding primary mouse brains. Using a ~2 h gradient on a 75 cm UPLC column with a modified data dependent acquisition method on a hybrid quadrupole-Orbitrap mass spectrometer (QE Plus), 5,062 total proteins were identified where 4,928 of those proteins were quantifiable by label-free quantitation (with 5 biological replicates). This analysis resulted in the most comprehensive proteomic dataset for ethanol- and LPS-treated primary mouse microglia to date and even expanded upon the well-characterized macrophage/microglia response to LPS treatment. This study also highlights the subtle, yet significant changes ethanol exposure can induce when compared to control. Interestingly, these changes are not consistent with the robust classical activation induced by LPS treatment, but instead align with the emerging theory that ethanol-treated microglia yield an alternative activation response. The contrast to LPS-treated microglia leads us to conclude that ethanol does not elicit a strong inflammatory response but rather might have a general inhibitory effect on multiple pathways such as phagocytosis and cell migration.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain, Microglial Cell

SUBMITTER: Stanley Stevens

LAB HEAD: Stanley Stevens

PROVIDER: PXD014466 | Pride | 2020-04-07

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	20180830_15sDDA_PriMg_50mM_EtOH_1.raw	Raw
	20180830_15sDDA_PriMg_50mM_EtOH_2.raw	Raw
	20180830_15sDDA_PriMg_50mM_EtOH_3.raw	Raw
	20180830_15sDDA_PriMg_50mM_EtOH_4.raw	Raw
	20180830_15sDDA_PriMg_50mM_EtOH_5.raw	Raw

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Publications

Deep proteome profiling reveals novel pathways associated with pro-inflammatory and alcohol-induced microglial activation phenotypes.

Guergues Jennifer J Wohlfahrt Jessica J Zhang Ping P Liu Bin B Stevens Stanley M SM

Journal of proteomics 20200318

Microglia, the resident immune cells of the brain, can exhibit a broad range of activation phenotypes, many of which have been implicated in several diseases and disorders of the central nervous system including those related to alcohol abuse. Given the complexity of global-scale molecular changes that define microglial activation, accurate phenotypic classification in the context of alcohol exposure is still lacking. We employed an optimized method for deep, quantitative proteome profiling of p ...[more]

PMID: 32200115

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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.