Unknown,Transcriptomics,Genomics,Proteomics

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Toward a Better Understanding of Potential Roles of Astrocytes in HIV-1-associated Neurocognitive Disorders

ABSTRACT: We present a microarray analysis of primary mouse astrocytes exposed to HIV-1 in culture. Results are compared with previous genomic studies of HIV-1 effect in human astrocytes and human and macaque brains. Day-two neonatal mouse astrocytes were exposed in triplicates to cell-free HIV-1/NL4-3 at m.o.i. of 1 and cultured for 24 hours. As control, cells were treated in triplicates with a mock virus concentrate at the same dilution (vol/vol) as the wild-type virus. As an example of large scale gene expression analysis we shall provide some details of the method; full description is in Kim et al. (S.-Y. Kim et al., 2004). Total RNA was prepared from cell cultures using the RNAeasy total RNA extraction kit (Qiagen, CA). RNA quality was assessed by electrophoresis and spectrophotometric analysis; between 1 and 10 µg of total RNA was used to generate a cDNA, and then 1 µg of cDNA product was used in an in vitro transcription reaction that contained biotinylated UTP and CTP. Twenty µg of full-length cRNA was fragmented and was subjected to gene expression analysis on the Affymetrix Mouse Genome 430 2.0 Array chip. Affymetrix software was used to generate CHP files. Significance analysis was performed using ArrayAssist software (Stratagene).

ORGANISM(S): Mus musculus

SUBMITTER: Alejandra Borjabad

PROVIDER: E-GEOD-17383 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Gene expression profiles of HIV-1-infected glia and brain: toward better understanding of the role of astrocytes in HIV-1-associated neurocognitive disorders.

Borjabad Alejandra A Brooks Andrew I AI Volsky David J DJ

Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology 20090821 1

Astrocytes are the major cellular component of the central nervous system (CNS), and they play multiple roles in brain development, normal brain function, and CNS responses to pathogens and injury. The functional versatility of astrocytes is linked to their ability to respond to a wide array of biological stimuli through finely orchestrated changes in cellular gene expression. Dysregulation of gene expression programs, generally by chronic exposure to pathogenic stimuli, may lead to dysfunction ...[more]

PMID: 19697136

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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.

Dataset Information

Toward a Better Understanding of Potential Roles of Astrocytes in HIV-1-associated Neurocognitive Disorders

Publications

Gene expression profiles of HIV-1-infected glia and brain: toward better understanding of the role of astrocytes in HIV-1-associated neurocognitive disorders.

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