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Characterization of MACS isolated microglia cells (CD11b positive cells) in comparison to all non-CD11b positive cells (termed non-target cell fraction (NTCF)) with proteomics

ABSTRACT: Microglia isolation from adult mouse brains remains difficult in comparison to microglia isolation from brains from newborn mouse brains as connective tissue, extracellular matrix materials and thicker myelin sheaths substantially influence traditional isolation protocols used with newborn mouse brains. Since the introduction of magnetic activated cell sorting (MACS), it has been reported that it is possible to also obtain microglia cells from adult mouse brains with a very good purity. However, most studies only used flow cytometry and/or qPCR to determine microglia enrichment and enrichment of traditional microglia marker proteins. Therefore we wanted to characterize in an unbiased way the proteomes of with MACS isolated microglia cells (CD11b positive cells) in comparison to all non-CD11b positive cells (termed non-target cell fraction (NTCF)), which consist of primarily astrocytes, neurons and oligodendrocytes. Therefore, we isolated from three adult mouse brains via MACS CD11b positive cells as well as the remaining CD11b negative cells (NTCF) to characterize the proteome of both cell fractions. The obtained proteomes were compared to a dataset of proteomic signatures for all CNS cell types determined by Sharma et al., (2015, doi: 10.1038/nn.4160) to determine the amount of enrichment of microglia-specific proteins.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Microglia

SUBMITTER: Emilio Cirri

LAB HEAD: Prof. Dr. med. habil. Reinhard Bauer

PROVIDER: PXD031973 | Pride | 2024-03-22

REPOSITORIES: Pride

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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.

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Characterization of MACS isolated microglia cells (CD11b positive cells) in comparison to all non-CD11b positive cells (termed non-target cell fraction (NTCF)) with proteomics

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