Dataset Information

Two-dimensional zymography differentiates gelatinase isoforms in stimulated microglial cells and in brain tissues of acute brain injuries.

ABSTRACT: Excessive activation of gelatinases (MMP-2/-9) is a key cause of detrimental outcomes in neurodegenerative diseases. A single-dimension zymography has been widely used to determine gelatinase expression and activity, but this method is inadequate in resolving complex enzyme isoforms, because gelatinase expression and activity could be modified at transcriptional and posttranslational levels. In this study, we investigated gelatinase isoforms under in vitro and in vivo conditions using two-dimensional (2D) gelatin zymography electrophoresis, a protocol allowing separation of proteins based on isoelectric points (pI) and molecular weights. We observed organomercuric chemical 4-aminophenylmercuric acetate-induced activation of MMP-2 isoforms with variant pI values in the conditioned medium of human fibrosarcoma HT1080 cells. Studies with murine BV-2 microglial cells indicated a series of proform MMP-9 spots separated by variant pI values due to stimulation with lipopolysaccharide (LPS). The MMP-9 pI values were shifted after treatment with alkaline phosphatase, suggesting presence of phosphorylated isoforms due to the proinflammatory stimulation. Similar MMP-9 isoforms with variant pI values in the same molecular weight were also found in mouse brains after ischemic and traumatic brain injuries. In contrast, there was no detectable pI differentiation of MMP-9 in the brains of chronic Zucker obese rats. These results demonstrated effective use of 2D zymography to separate modified MMP isoforms with variant pI values and to detect posttranslational modifications under different pathological conditions.

SUBMITTER: Chen S

PROVIDER: S-EPMC4393235 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Two-dimensional zymography differentiates gelatinase isoforms in stimulated microglial cells and in brain tissues of acute brain injuries.

Chen Shanyan S Meng Fanjun F Chen Zhenzhou Z Tomlinson Brittany N BN Wesley Jennifer M JM Sun Grace Y GY Whaley-Connell Adam T AT Sowers James R JR Cui Jiankun J Gu Zezong Z

PloS one 20150410 4

Excessive activation of gelatinases (MMP-2/-9) is a key cause of detrimental outcomes in neurodegenerative diseases. A single-dimension zymography has been widely used to determine gelatinase expression and activity, but this method is inadequate in resolving complex enzyme isoforms, because gelatinase expression and activity could be modified at transcriptional and posttranslational levels. In this study, we investigated gelatinase isoforms under in vitro and in vivo conditions using two-dimens ...[more]

PMID: 25859655

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Project description:Resident macrophages in the CNS microglia become activated and produce proinflammatory molecules upon encountering bacteria or viruses. TLRs are a phylogenetically conserved diverse family of sensors that drive innate immune responses following interactions with PAMPs. TLR3 and TLR4 recognize viral dsRNA Poly (I:C) and bacterial endotoxin LPS, respectively. Importantly, these receptors differ in their downstream adaptor molecules. Thus far, only a few studies have investigated the effects of TLR3 and TLR4 in macrophages. However, a genome-wide search for the effects of these TLRs has not been performed in microglia using RNA-seq. Gene expression patterns were determined for the BV-2 microglial cell line when stimulated with viral dsRNA Poly (I:C) or bacterial endotoxin LPS to identify novel transcribed genes, as well as investigate how differences in downstream signaling could influence gene expression in innate immunity.Sequencing assessment and quality evaluation revealed that common and unique patterns of proinflammatory genes were significantly up-regulated in response to TLR3 and TLR4 stimulation. However, the IFN/viral response gene showed a stronger response to TLR3 stimulation than to TLR4 stimulation. Unexpectedly, TLR3 and TLR4 stimulation did not activate IFN-ß and IRF3 in BV-2 microglia. Most importantly, we observed that previously unidentified transcription factors (TFs) (i.e., IRF1, IRF7, and IRF9) and the epigenetic regulators KDM4A and DNMT3L were significantly up-regulated in both TLR3- and TLR4-stimulated microglia. We also identified 29 previously unidentified genes that are important in immune regulation. In addition, we confirmed the expressions of key inflammatory genes as well as pro-inflammatory mediators in the supernatants were significantly induced in TLR3-and TLR4-stimulated primary microglial cells. Moreover, transcriptional start sites (TSSs) and isoforms, as well as differential promoter usage, revealed a complex pattern of transcriptional and post-transcriptional gene regulation upon infection with TLR3 and TLR4. Furthermore, TF motif analysis (-950 to +50 bp of the 5' upstream promoters) revealed that the DNA sequences for NF-?B, IRF1, and STAT1 were significantly enriched in TLR3- and TLR4-stimulated microglia.These unprecedented findings not only permit a comparison of TLR3-and TLR4-stimulated genes but also identify new genes that have not been previously implicated in innate immunity.

Project description:BackgroundBrain-resident microglia have a distinct origin compared to macrophages in other organs. Under physiological conditions, microglia are maintained by self-renewal from the local pool, independent of hematopoietic progenitors. Pharmacological depletion of microglia during whole-brain radiotherapy prevents synaptic loss and long-term recognition memory deficits. However, the origin or repopulated cells and the mechanisms behind these protective effects are unknown.MethodsCD45low/int/CD11b+ cells from naïve brains, irradiated brains, PLX5622-treated brains and PLX5622 + whole-brain radiotherapy-treated brains were FACS sorted and sequenced for transcriptomic comparisons. Bone marrow chimeras were used to trace the origin and long-term morphology of repopulated cells after PLX5622 and whole-brain radiotherapy. FACS analyses of intrinsic and exotic synaptic compartments were used to measure phagocytic activities of microglia and repopulated cells. In addition, concussive brain injuries were given to PLX5622 and brain-irradiated mice to study the potential protective functions of repopulated cells after PLX5622 + whole-brain radiotherapy.ResultsAfter a combination of whole-brain radiotherapy and microglia depletion, repopulated cells are brain-engrafted macrophages that originate from circulating monocytes. Comparisons of transcriptomes reveal that brain-engrafted macrophages have an intermediate phenotype that resembles both monocytes and embryonic microglia. In addition, brain-engrafted macrophages display reduced phagocytic activity for synaptic compartments compared to microglia from normal brains in response to a secondary concussive brain injury. Importantly, replacement of microglia by brain-engrafted macrophages spare mice from whole-brain radiotherapy-induced long-term cognitive deficits, and prevent concussive injury-induced memory loss.ConclusionsBrain-engrafted macrophages prevent radiation- and concussion-induced brain injuries and cognitive deficits.

Dataset Information

Two-dimensional zymography differentiates gelatinase isoforms in stimulated microglial cells and in brain tissues of acute brain injuries.

Publications

Two-dimensional zymography differentiates gelatinase isoforms in stimulated microglial cells and in brain tissues of acute brain injuries.

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