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

Guergues, Jennifer; Wohlfahrt, Jessica; Zhang, Ping; Liu, Bin; Stevens, Stanley M.

doi:10.1016/j.jprot.2020.103753

Cited by 18 publications

(29 citation statements)

References 61 publications

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“…Furthermore, the predominant majority of the significantly expressed proteins in the male mice were downregulated which was not the case for the female mice (Table S2). One common theme between both is the absence of canonical proinflammatory factors (Table S1), consistent with our previous studies using microglial cells treated EtOH (≤50 mM) (Bell-Temin et al, 2015;Bell-Temin et al, 2013;Guergues et al, 2020) and rat microglia following binge EtOH administration (Marshall et al, 2013). A significant result from the proteomic analysis was that the differentially expressed proteins show significant overlap as known downstream targets of several transcriptional and regulatory protein complexes.…”

Section: Sex-related Differential Responses Of Brain Microglia Followsupporting

confidence: 86%

“…The involvement of neuroimmune responses has been implicated in the pathophysiology of alcohol use disorders (Blednov et al, 2012;Karlsson et al, 2017). Various in vitro and animal studies have reported a spectrum of microglial phenotypes in responses to EtOH, ranging from classical proinflammatory (Pascual et al, 2011;Qin and Crews, 2012a;Qin and Crews, 2012b) to noninflammatory phenotypes (Bell-Temin et al, 2015;Bell-Temin et al, 2013;Guergues et al, 2020;Marshall et al, 2013) and a mix of both (Peng et al, 2017). Those differences are not fully understood, but variations in specifics of animal models, including EtOH exposure paradigm and dose, might be partially responsible.…”

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confidence: 99%

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Chronic Voluntary Binge Ethanol Consumption Causes Sex‐Specific Differences in Microglial Signaling Pathways and Withdrawal‐associated Behaviors in Mice

Rath

Guergues

Pinho

et al. 2020

Alcoholism Clin & Exp Res

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Background Microglia are the resident immune cells in the brain where they play essential roles in the development and maintenance of physiological functions of this organ. Aberrant activation of microglia is speculated to be involved in the pathogenesis of a variety of neurological disorders, including alcohol use disorders. Repeated binge ethanol (EtOH) consumption can have a profound impact on the function and integrity of the brain resulting in changes in behaviors such as withdrawal and reward. However, the microglial molecular and cellular pathways associated with EtOH binge consumption remain poorly understood. Method In this study, adult C57BL/6J male and female mice were subjected daily to a gelatin‐based drinking‐in‐the‐dark voluntary EtOH consumption paradigm (3 h/d for 4 months) to characterize EtOH consumption and withdrawal‐associated and anxiety‐like behaviors. Brain microglia were isolated at the end and analyzed for protein expression profile changes using unbiased mass spectrometry‐based proteomic analysis. Results Both male and female mice consistently consumed binge quantities of EtOH daily, resulting in blood EtOH levels > 80 mg/dl measured at the end of the 3‐hour daily consumption period. Although female mice consumed a significantly greater amount of EtOH than male mice, EtOH withdrawal‐associated anxiety‐like behaviors measured by marble‐burying, light‐dark box, and elevated plus maze tests were predominantly observed in male mice. Proteomic analysis of microglia isolated from the brains of animals at the end of the 4‐month binge EtOH consumption identified 117 and 37 proteins that were significantly up‐ or downregulated in EtOH‐exposed male and female mice, respectively, compared to their pair‐fed controls. Protein expression profile‐based pathway analysis identified several cellular pathways that may underlie the sex‐specific and EtOH withdrawal‐associated behavioral abnormalities. Conclusion Taken together, our findings revealed sex‐specific changes in EtOH withdrawal‐associated behaviors and signaling pathways in the mouse brain microglia and may help advance our understanding of the molecular, cellular, and behavioral changes related to human binge EtOH consumption.

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Section: Sex-related Differential Responses Of Brain Microglia Followsupporting

confidence: 86%

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confidence: 99%

Chronic Voluntary Binge Ethanol Consumption Causes Sex‐Specific Differences in Microglial Signaling Pathways and Withdrawal‐associated Behaviors in Mice

Rath

Guergues

Pinho

et al. 2020

Alcoholism Clin & Exp Res

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“…To further characterize global changes in an unbiased manner, we prepared experimental samples from Untreated (UNT), FAC-treated (FAC), control virus (CV), and oncogenic cocktail virus (OCV) infected FT194 cells; these were utilized for both noncoding RNA microarray and proteomic analyses. Specifically, the proteomic study utilized flash frozen cell pellets (UNT/FAC or CV/OCV) of ~ 500,000 cells per replicate, based on the protein extraction yield obtained by the S-trap sample processing approach previously reported by us 21 , 22 . For LFQ-based quantitation of protein expression, 5 replicates (from the same “batch” of UNT/FAC or CV/OCV cells) per group were utilized based on the expected quantitation precision of our approach obtained for cell lines 22 .…”

Section: Methodsmentioning

confidence: 99%

“…Digested samples were first concentrated on a 2 cm × 75 µm ID PepMap C18 trap column (Thermo Fisher Scientific) followed by separation on a 55 °C-heated, 75 cm × 75 µm ID C18 PepMap column (Thermo Fisher Scientific). A 120 min gradient from 2 to 28% B, where B was 0.1% formic acid in 80% acetonitrile:20% water was used to separate peptides, as described in our prior publication 21 . An additional ramp to 40% B over 15 min followed by a wash at 95% B was implemented.…”

Section: Methodsmentioning

confidence: 99%

Global miRNA/proteomic analyses identify miRNAs at 14q32 and 3p21, which contribute to features of chronic iron-exposed fallopian tube epithelial cells

Chhabra

Rockfield

Guergues

et al. 2021

Sci Rep

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Malignant transformation of fallopian tube secretory epithelial cells (FTSECs) is a key contributing event to the development of high-grade serous ovarian carcinoma (HGSOC). Our recent findings implicate oncogenic transformative events in chronic iron-exposed FTSECs, including increased expression of oncogenic mediators, increased telomerase transcripts, and increased growth/migratory potential. Herein, we extend these studies by implementing an integrated transcriptomic and mass spectrometry-based proteomics approach to identify global miRNA and protein alterations, for which we also investigate a subset of these targets to iron-induced functional alterations. Proteomic analysis identified > 4500 proteins, of which 243 targets were differentially expressed. Sixty-five differentially expressed miRNAs were identified, of which 35 were associated with the “top” proteomic molecules (> fourfold change) identified by Ingenuity Pathway Analysis. Twenty of these 35 miRNAs are at the 14q32 locus (encoding a cluster of 54 miRNAs) with potential to be regulated by DNA methylation and histone deacetylation. At 14q32, miR-432-5p and miR-127-3p were ~ 100-fold downregulated whereas miR-138-5p was 16-fold downregulated at 3p21 in chronic iron-exposed FTSECs. Combinatorial treatment with methyltransferase and deacetylation inhibitors reversed expression of these miRNAs, suggesting chronic iron exposure alters miRNA expression via epigenetic alterations. In addition, PAX8, an important target in HGSOC and a potential miRNA target (from IPA) was epigenetically deregulated in iron-exposed FTSECs. However, both PAX8 and ALDH1A2 (another IPA-predicted target) were experimentally identified to be independently regulated by these miRNAs although TERT RNA was partially regulated by miR-138-5p. Interestingly, overexpression of miR-432-5p diminished cell numbers induced by long-term iron exposure in FTSECs. Collectively, our global profiling approaches uncovered patterns of miRNA and proteomic alterations that may be regulated by genome-wide epigenetic alterations and contribute to functional alterations induced by chronic iron exposure in FTSECs. This study may provide a platform to identify future biomarkers for early ovarian cancer detection and new targets for therapy.

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“…Data. Raw proteomic data from a published study of mouse primary microglia 10 was downloaded from the Pride repository 11 (identifier PXD014466, https://www.ebi.ac.uk/pride/archive/projects/ PXD014466). The data set was composed of five biological replicates each from three sample groups: control, 50 mM ethanol treatment, or 5 ng lipopolysaccharide (LPS) treatment (15 total files).…”

Section: Methodsmentioning

confidence: 99%

Detection of Discordant Peptide Quantities in Shotgun Proteomics Data by Peptide Correlation Analysis (PeCorA)

Meyer

2020

Preprint

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Shotgun proteomics techniques infer the presence and quantity of proteins using peptide proxies, which are produced by cleavage of all isolated protein by a protease. Most protein quantitation strategies assume that multiple peptides derived from a protein will behave quantitatively similar across treatment groups, but this assumption may be false for biological or technical reasons. Here, I describe a strategy called peptide correlation analysis (PeCorA) that detects quantitative disagreements between peptides mapped to the same protein. Simple linear models are used to assess whether the slope of a peptide's change across treatment groups differs from the slope of all other peptides assigned to the same protein. Reanalysis of proteomic data from primary mouse microglia with PeCorA revealed that about 15% of proteins contain one discordant peptide. Inspection of the discordant peptides shows utility of PeCorA for direct and indirect detection of regulated PTMs, and also for discovery of poorly quantified peptides that should be excluded. PeCorA can be applied to an arbitrary list of quantified peptides, and is freely available as a script written in R.

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

Cited by 18 publications

References 61 publications

Chronic Voluntary Binge Ethanol Consumption Causes Sex‐Specific Differences in Microglial Signaling Pathways and Withdrawal‐associated Behaviors in Mice

Chronic Voluntary Binge Ethanol Consumption Causes Sex‐Specific Differences in Microglial Signaling Pathways and Withdrawal‐associated Behaviors in Mice

Global miRNA/proteomic analyses identify miRNAs at 14q32 and 3p21, which contribute to features of chronic iron-exposed fallopian tube epithelial cells

Detection of Discordant Peptide Quantities in Shotgun Proteomics Data by Peptide Correlation Analysis (PeCorA)

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