Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Gene expression in astrocytes upon sustained GSK-3 inhibition by small molecule

ABSTRACT: In order to identify molecular targets that mediates the stimulation of astrocyte's migration upon sustained specific inhibition of GSK-3, we have employed whole genome microarray expression profiling. Murine cortical-striatal astrocytes grown in primary culture were treated in vitro for 48 h with either 1 micromolar Ro3303544, or control medium (final equivalent concentration of DMSO), or washed-out for 48h in control medium after an initial 48h treatment with 1 micromolar Ro3303544. The three different conditions: Ctrl, Ro and wash-out (each in duplicate) were compared.

ORGANISM(S): Mus musculus

SUBMITTER: francois renault-mihara

PROVIDER: E-GEOD-25770 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Beneficial compaction of spinal cord lesion by migrating astrocytes through glycogen synthase kinase-3 inhibition.

Renault-Mihara Francois F Katoh Hiroyuki H Ikegami Takeshi T Iwanami Akio A Mukaino Masahiko M Yasuda Akimasa A Nori Satoshi S Mabuchi Yo Y Tada Hirobumi H Shibata Shinsuke S Saito Ken K Matsushita Masayuki M Kaibuchi Kozo K Okada Seiji S Toyama Yoshiaki Y Nakamura Masaya M Okano Hideyuki H

EMBO molecular medicine 20111018 11

The migratory response of astrocytes is essential for restricting inflammation and preserving tissue function after spinal cord injury (SCI), but the mechanisms involved are poorly understood. Here, we observed stimulation of in vitro astrocyte migration by the new potent glycogen synthase kinase-3 (GSK-3) inhibitor Ro3303544 and investigated the effect of Ro3303544 administration for 5 days following SCI in mice. This treatment resulted in accelerated migration of reactive astrocytes to sequest ...[more]

PMID: 21898827

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Project description:Research regarding the role of astrocytes as M-NM-2-amyloid (AM-NM-2) degrading cells has broadened our view about the mechanisms how these common glia cells function in AlzheimerM-bM-^@M-^Ys disease (AD). Based on previous studies adult mouse astrocytes are able to degrade AM-NM-2 deposits from AD mouse model and human brain tissue sections ex vivo, contrary to neonatal astrocytes. In this study, we studied the possible altered gene expression profiles of adult and neonatal astrocytes cultured for 22 h on top of the AM-NM-2 burdened tg APdE9 or wild-type mouse brain sections using whole genome microarrays. Quantitative RT-PCR analysis confirmed the significant up-regulation of HtrA serine peptidase 1 (Htra1), metallopeptidase 9 (Mmp9), phosphate regulating gene with homologies to endopeptidases on the X chromosome (Phex) and scavenger receptor class A, member 5 (Scara5) in adult astrocytes, whereas neonatal astrocytes up-regulated Mmp9 and down-regulated genes related to cholesterol transport and synthesis: apolipoprotein E (Apoe), 24-dehydrocholesterol reductase (Dhcr24) and 3-hydroxy-3-methylglutaryl-CoA synthase 1 (Hmgcs1). These findings brought out novel genes which expression is altered during AM-NM-2 clearance in adult and neonatal astrocytes ex vivo. Astrocyte cultures: For the adult astrocyte cultures, hippocampi and cortices were isolated from 6-8-week-old C57Bl/6j wild-type and tg eGFP mice and the tissue was suspended in DMEM/F12 (3:1, Gibco BRL, NY, USA) containing 10 % heat-inactivated fetal bovine serum (Gibco BRL) and 100 U / ml penicillin-streptomycin (Gibco BRL). The cells were treated with Percoll (Sigma-Aldrich, St.Louis, USA) and plated onto poly-L-lysine coated flasks in DMEM/F12 (3:1) containing 10 % heat-inactivated fetal bovine serum, 100 U / ml penicillin-streptomycin and G5 supplement (Gibco BRL). Before the experiments the glial cell cultures were shaken to remove microglia at 200 rpm for 2 h. For the neonatal astrocyte cultures, hippocampi and cortices were isolated from 2-day-old wild-type and eGFP mice according to the protocol described above, except that the culture medium was DMEM (+4500mg/l glucose, + L-glutamine, -pyruvate, Gibco BRL) without G5 supplement and the isolation method included no Percoll treatment. Standard anti-GFAP (1:500; Dako Cytomation, Glostrup, Denmark) immunocytochemistry was used to determine the purity of astrocyte cultures. The cells were incubated with biotinylated goat anti-rabbit IgG (1:200 dilution; Vector Laboratories, CA) secondary antibody and Vectastain ABC Elite kit was used (Vector Laboratories) according to manufacturerM-bM-^@M-^Ys protocol. Hydroxen peroxide (H2O2) and nickel enhanced diaminobenzidine (Ni-DAB) was added to detect the immunoreactivity. Three fields per well and at least three wells per cell type were analyzed for the percentage of Ni-DAB stained cells of all cellular profiles in the field. Adult and neonatal astrocyte cultures both contained on average 99.6 % M-BM-1 0.6 % anti-GFAP immunoreactive astrocytes. Anti-Iba (2 M-BM-5g/ml; Wako, Germany) and anti-CD11b (1:500 dilution; Serotec, UK) antibodies were used to detect possible microglial cells. The cultures contained less than 0.4 % of microglial cells in all experiments. Ex vivo treatments: tg APdE9 / wild-type mouse brain section incubated with neonatal (age 3 d) or adult (age 6-8 wks) mouse eGFP expressing astrocytes (4 x 105 cells / well), n=6. The mouse brain sections used were prepared from tg APdE9 mouse line (created by co-injection of chimeric mouse/human APPSwe and PS1-dE9 vectors, both controlled by their own mouse prion protein promoter element [APdE9 mice (Jankowsky et al. 2004) provided by Prof. Heikki Tanila] and their wild type C57Bl/6j littermates. Before the mRNA isolation process, the samples were pooled (n=3). The purity of the samples was checked with FACS (FACSCalibur, Becton Dickinson, USA) using the analyzing program CellQuestTM version 3.3 (Becton Dickinson, USA). After 22 h incubation the culture medium was removed from the wells and the brain sections with cultured eGFP astrocytes were incubated with 1 x trypsin-EDTA (500 M-BM-5l /well) for up to 15 min at 37M-BM-0 C. Partial detachment (at this point, the astrocytes should not lose the grip completely) of the astrocytes from the underlying brain section was monitored with a standard light microscope. After the incubation the brain sections with cultured astrocytes were carefully removed into small petri dishes filled with FBS-containing culture medium to inactivate the trypsin. Subsequently, the brain sections were immediately examined with fluorescence microscope (Olympus IX-71, Japan). The eGFP astrocytes were separated from the underlying brain section using a special suction hose, which was a modified version from suction hoses typically used for the microinjection techniques. In the other end of the tube there is a pipet tip as a mouthpiece, in the middle of the tube a 0.22 M-BM-5m filter and in the other end a stretched glass capillary (prepared with a Bunsen burner). The green fluorescent eGFP astrocytes were picked up gently from the underlying brain section (without breaking the brain tissue) using wavelength 468 nm of the fluorescence microscope and simultaneous light microscopy. Data normalization and analysis: The raw data was preprocessed with the robust multichip algorithm (Irizarry et al. 2003), normalized per chip to the median. 12 samples were grouped into 4 sample types: adult_wt, adult_tg, neonatal_wt, neonatal_tg. The change in the astrocyte gene expression was identified according to the sections the astrocytes were incubated (WT or TG). The differentially expressed probe sets were further processed using statistical analysis with Welch unpaired t-test assuming unequal variances (p<0.05; fold change > 2.0 or < 0.5). Benjamini-Hochberg false-discovery rate (FDR) correction for multiple testing was used to control the number of false-positives results.

Project description:Since macrophage activation can now be studied at a global level using modern microarray and proteomic analyses, discovery of novel macrophage activation genes is inevitable and important for understanding HIV-associated dementia (HAD). We isolated two different types of primary human macrophages, microglia and monocyte-derived macrophages (MDM) from brain tissue and whole blood respectively. The microarray analysis of differentially regulated macrophage activation genes reported here supports our previous assertions that the mixed glia (MIX) cultured in starvation conditions (DMEM alone) are a non-activated, or quiescent, tissue culture model for studying macrophage activation in the brain. Transcript levels from these quiescent cultures provided a background level of gene expression, and allowed for the identification of upregulated macrophage activation genes in the MIX brain cultures upon treatment with an array of soluble activation factors: serum components, cytokines, and growth factors. We found that 914 genes in the MIX cultures and 734 genes in the MDM cultures had a greater than 2-fold increase in expression. We discovered 180 genes that were increased greater than 2 fold in both culture types. Microarray-specific statistical analyses were performed to complement fold change analysis: Significance Analysis of Microarrays (SAM), and Partek Pro. In the MIX cultures, we detected over a hundred fold increase in IL-1? and TIMP1 transcription; Caspase 9, S100A8 and 9, MMP-12, IL-8, MCP-1, MRC-1, and IL-6 were also upregulated. Activation of starved MDM cultures resulted in fewer upregulated genes compared to MIX cultures. Genes upregulated in both MIX and MDM included CCL2 (MCP1), CCL7, CXCL5, TNFSF14, kinases, and phosphatases. These microarray data may provide leads for identifying previously unknown neurotoxins, disease biomarkers, and pathways responsible for the neuronal apoptosis observed in HAD, and for the eventual identification of therapeutic targets and treatments. For each gene, we performed a per-gene normalization, whereby an expression ratio was calculated by dividing the raw data from the activated media samples by the raw signal for the same gene from the non-activated media samples (Fig. 1). The same microarray data were also analyzed again using the very common per-chip normalization method recommended by the manufacturer where raw signals for each chip were multiplied by a scaling factor such that the trimmed mean for each chip became an arbitrary number (150) (Affymetrix, 2001). The per-chip normalization method reduces person-to-person biological variability and potentially hides important changes in gene expression (Hill et al., 2001),(Bolstad et al., 2003). Therefore, we report our data using a per-gene normalization method, which yielded higher signal and low noise (uninteresting non-biological variability caused by chips, and/or experimental sample preparation), and given our experimental design, reduced person-to-person variability that these experiments cannot address directly. For later statistical analyses, the data were log transformed in order to obtain a normal distribution of the expression values of the >12,625 genes (Bolstad et al., 2003)

Dataset Information

Gene expression in astrocytes upon sustained GSK-3 inhibition by small molecule

Publications

Beneficial compaction of spinal cord lesion by migrating astrocytes through glycogen synthase kinase-3 inhibition.

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