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Quantitative Proteomics of Acutely Isolated Mouse Microglia Identifies Novel Immune Alzheimer's Disease-related Proteins

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Microglia

DISEASE(S): Alzheimer's Disease

SUBMITTER: Eric Dammer

LAB HEAD: Nicholas T. Seyfried

PROVIDER: PXD009137 | Pride | 2018-09-18

REPOSITORIES: Pride

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Quantitative proteomics of acutely-isolated mouse microglia identifies novel immune Alzheimer's disease-related proteins.

Rangaraju Srikant S Dammer Eric B EB Raza Syed Ali SA Gao Tianwen T Xiao Hailian H Betarbet Ranjita R Duong Duc M DM Webster James A JA Hales Chadwick M CM Lah James J JJ Levey Allan I AI Seyfried Nicholas T NT

Molecular neurodegeneration 20180628 1

<h4>Background</h4>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 spectrometry methods can comprehensively characterize proteomic alterations in microglia in neurodegenerative disorders, potentially providing novel function ...[more]

PMID: 29954413

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Project description:Alzheimer’s Disease (AD) is the most common cause of dementia in the elderly population without a cure or early diagnostics currently available. Despite the demonstrated ability of extracellular vesicles (EVs) to spread tau and Aβ pathology in AD models and their potential utility as a diagnostic and treatment-monitoring tool, our knowledge of human brain EV subpopulations, their molecular composition and roles in disease progression is very limited. Genome-wide association studies linked multiple AD genetic risk factors to microglia-specific pathways suggesting a potential role of microglia-derived EVs in AD progression. Here we are presenting a method for isolation of microglial CD11b-positive small EVs from cryopreserved human brain tissue and multi-omics analysis of the EVs from the parietal cortex of 4 late-stage AD (Braak V-VI) and 3 age-matched normal/low pathology (NL) cases. We identified a total of 1000 proteins by quantitative proteomics, analyzed 594 individual lipid species by targeted lipidomics, and 105 miRNAs using a NanoString miRNA expression panel. We found significant reduction in abundance of homeostatic microglia markers, P2RY12 and TMEM119, and increased levels of disease associated microglia markers, FTH1 and TREM2, in microglial EVs from AD brain compared to NL cases. Protein tau abundance was also significantly higher in AD brain-derived microglial EVs. These changes were accompanied by upregulation of synaptic and neuron-specific proteins in the AD group. Levels of free-cholesterol were elevated in microglial EVs from AD brain. Lipidomic analysis also revealed a pro-inflammatory lipid profile, endolysosomal dysfunction and significant AD-associated decrease in levels of docosahexaenoic acid (DHA)-containing polyunsaturated lipids of different classes, suggesting a potential defect in acyl-chain remodeling. Several immune pathways and senescence were among the top pathways controlled by 4 miRNAs significantly upregulated in the AD group. Our data suggest that loss of the homeostatic microglia signature in late AD stages may accompany endolysosomal impairment and release of undigested neuronal and myelin debris, including tau, through extracellular vesicles. These data validate a method of isolation of small cell-type specific EVs from human brain tissue, suggests new potential EV-associated markers and disease-related pathways, and provides a framework for future large-scale multi-omics study.

Project description:GFAP and vimentin deficiency alters gene expression in astrocytes and microglia in wild-type mice and changes the transcriptional response of reactive glia in mouse model for Alzheimer's disease. Reactive astrocytes with an increased expression of intermediate filament (IF) proteins Glial Fibrillary Acidic Protein (GFAP) and Vimentin (VIM) surround amyloid plaques in Alzheimer's disease (AD). The functional consequences of this upregulation are unclear. To identify molecular pathways coupled to IF regulation in reactive astrocytes, and to study the interaction with microglia, we examined WT and APPswe/PS1dE9 (AD) mice lacking either GFAP, or both VIM and GFAP, and determined the transcriptome of cortical astrocytes and microglia from 15- to 18-month-old mice. Genes involved in lysosomal degradation (including several cathepsins) and in inflammatory response (including Cxcl5, Tlr6, Tnf, Il1b) exhibited a higher AD-induced increase when GFAP, or VIM and GFAP, were absent. The expression of Aqp4 and Gja1 displayed the same pattern. The downregulation of neuronal support genes in astrocytes from AD mice was absent in GFAP/VIM null mice. In contrast, the absence of IFs did not affect the transcriptional alterations induced by AD in microglia, nor was the cortical plaque load altered. Visualizing astrocyte morphology in GFAP-eGFP mice showed no clear structural differences in GFAP/VIM null mice, but did show diminished interaction of astrocyte processes with plaques. Microglial proliferation increased similarly in all AD groups. In conclusion, absence of GFAP, or both GFAP and VIM, alters AD-induced changes in gene expression profile of astrocytes, showing a compensation of the decrease of neuronal support genes and a trend for a slightly higher inflammatory expression profile. However, this has no consequences for the development of plaque load, microglial proliferation, or microglial activation. 2 cell types from 6 conditions: cortical microglia and cortical astrocytes from 15-18 month old APPswe/PS1dE9 mice compared to wildtype littermates. Biological replicates: microglia from APPswe/PS1dE9, N=7, microglia from WT, N=7, astrocytes from APPswe/PS1dE9, N=4, microglia from WT, N=4

Dataset Information

Quantitative Proteomics of Acutely Isolated Mouse Microglia Identifies Novel Immune Alzheimer's Disease-related Proteins

Publications

Quantitative proteomics of acutely-isolated mouse microglia identifies novel immune Alzheimer's disease-related proteins.

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